Cloning and production of human von willebrand factor GPIb binding domain polypeptides and methods of using same

ABSTRACT

The subject invention provides non-glycosylated, biologically active polypeptides which comprise the vWF (von Willebrand Factor) GP1b binding domain. These polypeptides may be used to inhibit platelet adhesion and aggregation in the treatment of subjects with conditions such as cerebrovascular disorders and cardiovascular disorders. This invention also provides expression plasmids encoding these polypeptides as well as methods of producing by transforming a bacterial cell and recovering such polypeptides. In addition, the subject invention provides methods of treating and preventing cerebrovascular, cardiovascular and other disorders using these polypeptides to inhibit platelet aggregation.

This application is a continuation of U.S. Ser. No. 08/080,690, filedJun. 22, 1993, now abandoned; which is a continuation of U.S. Ser. No.07/753,815, filed Sep. 3, 1991, now abandoned; which is filed under 35USC 371 as the national stage of International Application No.PCT/US91/01416, filed Mar. 1, 1991; which is a continuation-in-part ofU.S. Ser. No. 07/487,767, filed Mar. 2, 1990, now abandoned, thecontents of which are hereby incorporated by reference into the presentdisclosure.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

This invention relates to the cloning and production of human vonWillebrand Factor analogs and methods of using such analogs.

Structural Features of von Willebrand Factor

Von Willebrand Factor (vWF) is a large plasma protein which issynthesized in the endothelial cells which form the inner surface liningof the blood vessel wall, and by megakarocytes, the precursor ofplatelets. Large amounts of vWF are found in platelet α-granules, whosecontents are released into the blood upon platelet activation. Newlysynthesized vWF in endothelial cells may enter the blood via twoalternative pathways. Part is secreted constitutively into the blood,mainly as disulfide-linked dimers or small multimers of a 250,000 daltonsubunit. Alternatively, part enters secretory organelles calledWeibel-Palade bodies. The vWF stored in Weibel-Palade bodies is highlymultimeric, ranging in size from that of a dimer to multimers of 50 ormore subunits, and can be released from the cells by treatment withsecretatogues, such as thrombin. The highly multimeric vWF is the mosteffective in promoting platelet adhesion.

The gene encoding vWF has been isolated and shown to be greater than 150kb in length. It is composed of over 20 exons. The vWF mRNA isapproximately 9000 bases in length and encodes a pre-pro-vWF of 2813amino acids. Residues 1-22 form a processed leader sequence whichpresumably is cleaved after entry of the protein into the roughendoplasmic reticulum. The N-terminal portion of the pro-vWF (741 aminoacids) is the pro-peptide which is not present in mature vWF. Thispeptide is present in the blood and has been shown to be identical to ablood protein previously known as von Willebrand Antigen II (vW AgII).The pro-peptide is essential for the multimerization of vWF. Cells intowhich a vWF cDNA containing only mature vWF sequences have beenintroduced produce only dimers. No function is known for thepropeptide/vW AgII.

DNA sequence analysis has demonstrated that the pro-vWF precursor iscomposed of repeated domain subunits. Four different domains have beenidentified. Mature vWF consists of three A type, three B type, and two Ctype domains. There are also two complete and one partial D type domain.The pro-peptide consists of two D type domains, leading to thespeculation that it may have associated functions.

Mature vWF is a multivalent molecule which has binding sites for severalproteins. One of the binding sites recognizes the platelet glycoproteinIb (GPIb). Using proteolytic digests this site has been localized to theregion between amino acid residues 449 and 728 of mature vWF. Inaddition, vWF has at least two collagen binding sites, at least twoheparin binding sites, a Factor VIII binding site, and a RGD site whichbinds to the platelet GP IIb/IIIa receptor.

Involvement Of vWF In Platelet Adhesion To Subendothelium

Evidence that vWF, and specifically, the binding of vWF to the plateletGPIb receptor, is essential for normal platelet adhesion, is based onboth clinical observations and in vitro studies. Patients with thebleeding disorder von Willebrand Disease (vWD) have reduced levels ofvWF or are completely lacking in vWF. Alternatively, they may havedefective vWF. Another disorder, Bernard-Soulier Syndrome (BSS), ischaracterized by platelets lacking GPIb receptors.

The in vitro system which most closely approximates the environment of adamaged blood vessel consists of a perfusion chamber in which an evertedblood vessel segment (rabbit aorta, human post-mortem renal artery, orthe human umbilical artery) is exposed to flowing blood. After strippingoff the layer of endothelial cells from the vessel, blood is allowed toflow through the chamber. The extent of platelet adhesion is estimateddirectly by morphometry or indirectly using radiolabeled platelets.Blood from patients with VWD or BSS does not support platelet adhesionin this system while normal blood does, indicating the need for vWF andplatelet GPIb. Moreover, addition of monoclonal antibodies to GPIbprevents platelet adhesion as well. The vWF-dependence of plateletadhesion is more pronounced under conditions of high shear rates, suchas that present in arterial flow. Under conditions of low shear rates,platelet adhesion may be facilitated by other adhesion proteins, such asfibronectin. Possibly, the adhesive forces provided by these otherproteins are not adequate to support adhesion at high shear forces, andvWF dependence becomes apparent. Also, the multimeric nature of the vWFmay provide for a stronger bond by binding more sites on the platelet.

About 20% of patients from whom clots have been removed by angioplastyor by administration of tissue plasminogen activator (tPA) sufferre-occlusion. This is presumably the result of damage to the endotheliumduring the treatment which results in the adhesion of platelets to theaffected region on the inner surface of the vessel. This is followed bythe aggregation of many layers of platelets and fibrin onto thepreviously adhered platelets, forming a thrombus.

To date none of the anti-platelet aggregation agents described in theliterature prevent the initial platelet adhesion to the exposedsub-endothelium thereby preventing subsequent clot formation.

The subject invention provides non-glycosylated, biologically activepolypeptides which comprise the vWF (von Willebrand Factor) GP1b bindingdomain. These polypeptides may be used inter alia to inhibit plateletadhesion and aggregation in the treatment of subjects with conditionssuch as cerebrovascular disorders and cardiovascular disorders. Thisinvention also provides expression plasmids encoding these polypeptidesas well as methods of producing by transforming a bacterial cell andrecovering such polypeptides. In addition, the subject inventionprovides methods of treating and preventing cerebrovascular,cardiovascular and other disorders using these polypeptides to inhibitplatelet aggregation.

SUMMARY OF THE INVENTION

This invention provides a non-glycosylated, biologically activepolypeptide having the amino acid sequence: ##STR1## wherein X is NH₂-methionine- or NH₂ --;

A is a sequence of at least 1, but less than 35 amino acids, whichsequence is present in naturally occurring vWF, the carboxy terminalamino acid of which is the tyrosine #508 shown in FIG. 12 SEQ ID NO.1;

B is a sequence of at least 1, but less than 211 amino acids, whichsequence is present in naturally occurring vWF, the amino terminal aminoacid of which is the aspartic acid #696 shown in FIG. 12 SEQ ID NO.1;and

the two cysteines included within the bracketed sequence are joined by adisulfide bond.

In addition, the subject invention provides a method of producing any ofthe above-described polypeptides which comprises transforming abacterial cell with an expression plasmid encoding the polypeptide,culturing the resulting bacterial cell so that the cell produces thepolypeptide encoded by the plasmid, and recovering the polypeptide soproduced.

Furthermore, the subject invention provides a pharmaceutical compositioncomprising an amount of any of the above-described polypeptideseffective to inhibit platelet aggregation and a pharmaceuticallyacceptable carrier. The subject invention also provides a method ofinhibiting platelet aggregation which comprises contacting plateletswith an amount of any of the above-described polypeptides effective toinhibit platelet aggregation. In addition, the subject inventionprovides methods of treating, preventing or inhibiting disorders such ascerebrovascular or cardiovascular disorders or thrombosis, comprisingadministering to the subject an amount of any of the above-describedpolypeptides effective to treat or prevent such disorders.

The subject invention also provides a method for recovering a purified,biologically active above-described polypeptide which comprises:

(a) producing in a bacterial cell a first polypeptide having the aminoacid sequence of the polypeptide but lacking the disulfide bond;

(b) disrupting the bacterial cell so as to produce a lysate containingthe first polypeptide;

(c) treating the lysate so as to obtain inclusion bodies containing thefirst polypeptide;

(d) contacting the inclusion bodies from step (c) so as to obtain thefirst polypeptide in soluble form;

(e) treating the resulting first polypeptide so as to form thebiologically active polypeptide;

(f) recovering the biologically active polypeptide so formed; and

(g) purifying the biologically active polypeptide so recovered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Construction of pvW1P

This figure shows the construction of plasmid pvW1P. A series of vWFcDNA clones in λ gt11 (isolated from a human endothelial cell cDNAlibrary) were isolated. One cDNA clone covering the entire GPIb bindingdomain was subcloned into the EcoRI site of pUC19. The resultingplasmid, pvW1P, contains a 2.5 kb cDNA insert.

FIG. 2: Construction of pvWF-VA1

This figure shows the construction of plasmid pvWF-VA1. A syntheticoligomer containing an ATG initiation codon located before the aminoacid glu-437 (i.e., the 437th amino acid in the vWF protein shown inFIG. 12) was ligated to plasmid pvW1P digested with NdeI and Bsu36I. Theresulting plasmid was designated pvWF-VA1, and has been deposited in E.coli strain Sφ930 under ATCC Accession No. 68530.

FIG. 3: Construction of pvWF-VB1

This figure shows the construction of plasmid pvWF-VB1. A syntheticoligomer containing an ATG initiation codon located before the aminoacid phe-443 (see FIG. 12) was ligated to plasmid pvW1p digested withNdeI and Bsu36I. The resulting plasmid was designated pvWF-VB1.

FIG. 4: Construction of pvWF-VA2

This figure shows the construction of plasmid pvWF-VA2. A syntheticoligomer containing a TAA termination codon located after the amino acidlys-728 (see FIG. 12) was ligated to plasmid pvWF-VA1 digested withHindIII and XmaI.

The resulting plasmid was designated pvWF-VA2.

FIG. 5: Construction of pvWF-VB2

This figure shows the construction of plasmid pvWF-VB2. A syntheticoligomer containing a TAA termination codon was ligated to plasmidpvWF-VB1 digested with HindIII and XmaI. The resulting plasmid wasdesignated pvWF-VB2.

FIG. 6: Construction of pvWF-VA3

This figure shows the construction of plasmid pvWF-VA3. An NdeI-EcoRVfragment was isolated from plasmid pvWF-VA2 and ligated to plasmidpMF-945 (constructed as described in FIG. 11) digested with NdeI andPvuII. The plasmid obtained was designated pvWF-VA3. The plasmidexpresses VA, a vWF GPIb binding domain polypeptide which includes aminoacids 437 to 728 (see FIG. 12) under the control of the deo P₁ P₂promoter.

FIG. 7: Construction of pvWF-VB3

This figure shows the construction of plasmid pvWF-VB3. An NdeI-EcoRVfragment was isolated from plasmid pvWF-VB2 and ligated to plasmidpMF-945 digested with NdeI and PvuII. The plasmid obtained wasdesignated pvWF-VB3. The plasmid expresses VB, a vWF GPIb binding domainpolypeptide which includes amino acids 443 to 728 under the control ofthe deo P₁ P₂ promoter.

FIG. 8: Construction of pvWF-VC3

This figure shows the construction of plasmid pvWF-VC3. A syntheticlinker was ligated to pvWF-VA3 digested with NdeI and Tth111I. Theplasmid obtained was designated pvWF-VC3, and has been deposited withthe ATCC under ATCC Accession No. 68241. The plasmid expresses VC (alsoreferred to as VCL or VC3), a vWF GPIb binding domain polypeptide whichincludes amino acids 504 to 728 (see FIG. 12) under the control of thedeo P₁ P₂ promoter.

FIG. 9: Construction of pvWF-VD3

This figure shows the construction of plasmid pvWF-VD3. A syntheticlinker was ligated to pvWF-VA3 digested with NdeI and Tth111I. Theplasmid obtained was designated pvWF-VD3. The plasmid expresses VD, avWF GPIb binding domain polypeptide which includes amino acids 513 to728 (see FIG. 12) under the control of the deo P₁ P₂ promoter.

FIG. 10: Relative Alignment of Plasmids Expressing vWF-GPIb BindingDomain Polypeptides

This figure shows the relative alignment of the plasmids expressing thevWF-GPIb binding domain polypeptides. Also shown on the top two linesare representations of the vWF cDNA and the location of the GPIb bindingdomain coding region within the cDNA.

FIG. 11: Construction of Plasmid pMF-945

This figure shows the construction of plasmid pMF-945. Plasmid pEFF-920(in Escherichia coli Sφ930, ATCC Accession No. 67706) was cleaved withBglII and NdeI, and the large fragment was isolated. This fragment wasligated to the small 540 bp fragment produced by cleaving plasmidpMF-5534 (ATCC Accession No. 67703) with BglII and NdeI. This producesplasmid pMF-945 which harbors the PAR sequence and in 5' and 3' orderthe deo P₁ P₂ promoter sequences, the modified deo ribosomal bindingsite with an enhancer sequence, a pGH analog coding sequence and the T₁T₂ transcription termination sequences. Plasmid pMF-945 is a high levelexpressor of pGH analog protein.

FIG. 12: Translated cDNA Sequence of Mature Human vWF

This figure which consists of FIGS. 12A, 12B, 12C, 12D, 12E, 12F, 12Gand 12H shows the translated cDNA sequence of mature human vonWillebrand Factor SEQ ID NO.1.

This sequence was compiled using the data disclosed by Verweij, C. L.,et al., EMBO Journal 5: 1839-1847 (1986) and Sadler, J. E., et al.,Proc. Natl. Acad. Sci. 82: 6394-6398 (1985). This nucleotide sequencecommences with nucleotide number 2519 (where nucleotide 1 relates to thestart of the coding sequence for the signal peptide) and terminates withnucleotide 8668, a total of 6150 nucleotides encoding mature vWFconsisting of 2050 amino acids. The translated amino acid sequencecommences with amino acid number 1 and terminates with amino acid number2050. The corresponding nucleotide and amino acid designations are usedthroughout this application.

FIG. 13: Translated Sequence of VC, the vWF GPIb Binding DomainPolypeptide Expressed by Plasmids pvWF-VC3 and pvWF-VCL

This figure shows the translated sequence of the von Willebrand FactorGPIb binding domain polypeptide expressed by plasmids pvWF-VC3 (ATCCAccession No. 68241) and pvWF-VCL (ATCC Accession No. 68242 SEQ IDNO.2).

The first codon ATG encoding the translation initiation codon methioninehas been added to the nucleotide sequence corresponding to nucleotides4028 to 4702 of the sequence of FIG. 12. This sequence encodes apolypeptide containing 225 amino acids (plus the initiation methionine)corresponding to amino acid Leu 504 to amino acid Lys 728 of FIG. 12,i.e. 226 amino acids in total.

FIG. 14: Construction of pvWF-VCL

This figure shows the construction of plasmid pvWF-VCL. Plasmid pvWF-VC3was digested with HindIII and StyI and the 860 base pair fragmentisolated. This fragment was ligated with the large fragment isolatedfrom the HindIII-StyI digest of plasmid pMLK-100. The resulting plasmidwas designated pvWF-VCL and deposited in E. coli 4300(F⁻) with the ATCCunder ATCC Accession No. 68242. This plasmid expresses VCL, the same vWFGPIb binding domain polypeptide as pvWF-VC3 (methionine plus amino acids504-728), however under control of the λP_(L) promoter and the deoribosomal binding site.

FIG. 15: Construction of Plasmid pvWF-VE2

Plasmid pvWF-VA2 was digested with NdeI and PstI and the large fragmentisolated. Synthetic oligomers No. 2 and No. 3 (shown in FIG. 16) weretreated with T4 polynucleotide kinase. The large pvWF-VA2 fragment wasthen ligated with synthetic oligomers No. 1 and No. 4, (shown in FIG.16) and with kinased oligomers No. 2 and No. 3. The resulting plasmidwas designated pvWF-VE2.

FIG. 16: Synthetic Oligomers Used in Construction of pvWF-VE2.

This figure shows the four synthetic linkers (Nos. 1-4) used inconstruction of pvWF-VE2.

FIG. 17: Construction of Plasmid pvWF-VE3

Plasmid pvWF-VE2 was digested with NdeI and HindIII and the small 770 bpfragment isolated and ligated with the large fragment isolated from theNdeI-HindIII digest of plasmid pMLK-7891. The resulting plasmid wasdesignated pvWF-VE3.

FIG. 18: Construction of Plasmid pvWF-VEL

Plasmid pvWF-VE3 was digested with XmnI, treated with bacterial alkalinephosphatase (BAP), and further digested with NdeI and HindIII. PlasmidpMLK-100 was digested with NdeI and HindIII and treated with BAP. Thetwo digests were mixed and ligated, producing plasmid pvWF-VEL whichexpresses the DNA sequence corresponding to amino acids 469-728 ofmature vWF under the control of the λP_(L) promoter and the cIIribosomal binding site.

FIG. 19: The Effect of VCL on BJV-Induced Aggregation in Human PlateletRich Plasma (PRP)

This figure provides the results of a standardized von Willebrand Factor(vWF)-dependent aggregation assay using human PRP.

FIG. 20: The Effect of VCL on BJV-Induced Aggregation in Rat PRP

This figure provides the results of a standardized von Willebrand Factor(vWF)-dependent aggregation assay using rat PRP.

FIG. 21: Inhibition of Platelet Adhesion to Endothelial CellExtracellular Matrix (ECM) by the GPIb Binding Domain Polypeptide VCL

This figure shows the effect of VCL on binding of platelets toimmobilized ECM as described in Example 9. At both low and high shearrates, VCL is effective in reducing platelet adhesion to ECM in vitro.

FIG. 22: Dose Response Curve of Inhibition of Platelet Adhesion toEndothelial Cell Extracellular Matrix (ECM) by the GPIb Binding DomainPolypeptide VCL

A series of concentrations of VCL was tested to determine the IC₅₀ ofVCL in vitro as described in Example 9. As seen in the figure, the IC₅₀is about 0.8 μM VCL, while maximal inhibition is achieved at about 2 μMVCL.

FIG. 23: Inhibition of Platelet Adhesion to Fibrinogen by the GPIbBinding Domain Polypeptide VCL

Platelet adhesion to immobilized fibrinogen and laminin were tested inthe glass cover slip model as described in Example 9. VCL was moreinhibitory of platelet adhesion to fibrinogen at a high shear rate thanat a low shear rate. VCL did not inhibit binding of platelets tolaminin.

FIG. 24: Inhibition of Platelet Adhesion to Collagen, vWF, andFibronectin by the GPIb Binding Domain Polypeptide VCL

Platelet adhesion to immobilized collagen (Type I), immobilized vWF, andimmobilized fibronectin was tested in the model as described in Example9.

FIG. 25: Platelet Adhesion to VCL

VCL was also tested as a substrate in the model described in Example 9.Binding of platelets is low at both low and high shear rates (≦5%coverage).

DETAILED DESCRIPTION OF THE INVENTION

The plasmids pvWF-VC3, pvWF-VCL and pvWF-VA1 were deposited inEscherichia coli pursuant to, and in satisfaction of, the requirementsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852 under ATCC Accession Nos. 68241, 68242 and 68530, respectively.

This invention provides a non-glycosylated, biologically activepolypeptide having the amino acid sequence: ##STR2## wherein X is NH₂-methionine- or NH₂ --;

A is a sequence of at least 1, but less than 35 amino acids, whichsequence is present in naturally occurring human vWF, the carboxyterminal amino acid of which is the tyrosine #508 shown in FIG. 12 SEQID NO.1;

B is a sequence of at least 1, but less than 211 amino acids, whichsequence is present in naturally occurring human vWF, the amino terminalamino acid of which is the aspartic acid #696 shown in FIG. 12 SEQ IDNO.1; and the two cysteines included within the bracketed sequence arejoined by a disulfide bond. The bracketed sequence comprises amino acids#509-#695 of FIG. 12.

In one embodiment, this polypeptide has the amino acid sequence:##STR3## wherein X is NH₂ -- or NH₂ -methionine-, preferably NH₂-methionine-.

The bracketed sequence comprises amino acids #504-#728 of FIG. 12 SEQ IDNO.1.

One skilled in the art to which the subject invention pertains canreadily make such polypeptides using recombinant or non-recombinant DNAtechniques.

The polypeptides may be constructed using recombinant DNA technology.One means for obtaining the polypeptides is to express nucleic acidencoding the polypeptides in a suitable host, such as a bacterial,yeast, or mammalian cell, using methods well known in the art, andrecovering the polypeptide after it has been expressed in such a host.

Examples of vectors that may be used to express the nucleic acidencoding the polypeptides are viruses such as bacteriophages (such asphage lambda), cosmids, plasmids, and other recombination vectors.Nucleic acid molecules are inserted into vector genomes by methods wellknown in the art. For example, using conventional restriction enzymesites, insert and vector DNA can both be exposed to a restriction enzymeto create complementary ends on both molecules which base pair with eachother and are then ligated together with a ligase. Alternatively,linkers can be ligated to the insert DNA which correspond to arestriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable.

Vectors comprising nucleic acid encoding the polypeptides may be adaptedfor expression in a bacterial cell, a yeast cell, or a mammalian cellwhich additionally comprise the regulatory elements necessary forexpression of the nucleic acid in the bacterial, yeast, or mammaliancells so located relative to the nucleic acid encoding the polypeptideas to permit expression thereof. Regulatory elements required forexpression include promoter sequences to bind RNA polymerase andtranscription initiation sequences for ribosome binding.

For example, a bacterial expression vector may include a promoter suchas the λP_(L) or deo promoters and for transcription initiation theC_(II) or deo ribosomal binding sites. Such vectors may be obtainedcommercially or assembled from the sequences described by methods wellknown in the art, for example the methods described above forconstructing vectors in general.

In addition, non-recombinant techniques such as chemical synthesis,synthetic DNA or cDNA may be used to obtain the above-describedpolypeptides. One means of isolating the polypeptide is to probe a humangenomic library with a natural or artificially designed DNA probe, usingmethods well known in the art. DNA and cDNA molecules which encode thepolypeptides may be used to obtain complementary genomic DNA, cDNA orRNA from human, mammalian or other animal sources, or to isolate relatedcDNA or genomic clones by the screening of cDNA or genomic libraries.

The subject invention further provides a pharmaceutical compositioncomprising an amount of any of the above-described polypeptideseffective to inhibit platelet aggregation and a pharmaceuticallyacceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier"encompasses any of the standard pharmaceutical carriers. Such carriersare well known in the art and may include, but are in no way and are notintended to be limited to, any of the standard pharmaceutical carrierssuch as phosphate buffered saline solutions, water, emulsions such asoil/water emulsion, and various types of wetting agents. Other carriersmay also include sterile solutions, tablets, coated tablets, andcapsules.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods.

The composition has an amount sufficient to result in a bloodconcentration of 0.06 to 58 μM, preferably between about 0.06 and 29 μM,for example 0.23 to 23 μM. Expressed in different terms, the amountshould be 0.1 to 100 mg/Kg body weight, preferably 0.1 to 50 mg/Kg bodyweight, for example 0.4 to 40 mg/kG body weight.

The administration of the composition may be effected by any of the wellknown methods, including but not limited to intravenous, intramuscular,subcutaneous and oral administration.

This invention also provides a method of inhibiting platelet aggregationwhich comprises contacting platelets with an amount of any of theabove-described polypeptides effective to inhibit platelet aggregationso as to inhibit platelet aggregation. The range of the amount effectiveto inhibit platelet aggregation is 0.1-200 mg/kg body weight, preferably1-20 mg/kg body weight. The amount effective to inhibit plateletaggregation is an amount sufficient to maintain a blood concentration of0.1-10 μM polypeptide. In a preferred embodiment, the bloodconcentration is maintained at about 1 μM polypeptide.

This invention also provides expression plasmids encoding theabove-described polypeptides. In one embodiment, the expression plasmidencoding the polypeptide with the bracketed sequence, i.e. amino acids#504-#728 of FIG. 12, is designated pvWF-VC3 and is deposited under ATCCAccession No. 68241. In another embodiment, the expression plasmidencoding a polypeptide with the bracketed sequence, i.e. amino acids#504-#728 of FIG. 12, is designated pvWF-VCL and is deposited under ATCCAccession No. 68242.

The expression plasmids of this invention further comprise suitableregulatory elements positioned within the plasmid relative to the DNAencoding the polypeptide so as to effect expression of the polypeptidein a suitable host cell, such as promoter and operators, e.g. deo P₁ P₂and λP_(L) O_(L), ribosomal binding sites, e.g. deo and C_(II), andrepressors. Other suitable regulatory elements include, for example, thelac, trp, tac, and lpp promoters (European Patent ApplicationPublication No. 0303972, published Feb. 22, 1989).

The suitable regulatory elements are positioned within the plasmidrelative to the DNA encoding the polypeptide so as to effect expressionof the polypeptide in a suitable host cell. In preferred embodiments ofthe invention, the regulatory elements are positioned close to andupstream of the DNA encoding the polypeptide.

The expression plasmids of this invention may be introduced intosuitable host cells, preferably bacterial host cells. Preferredbacterial host cells are Escherichia coli cells. Examples of suitableEscherichia coli cells are strains Sφ930 or 4300, but other Escherichiacoli strains and other bacteria can also be used as host cells for theplasmids. Such bacteria include Pseudomonas aeruginosa and Bacillussubtilis.

The bacteria used as hosts may be any strain including auxotrophic (suchas A1645), prototrophic (such as A4255), and lytic strains; F⁺ and F⁻strains; strains harboring the cI⁸⁵⁷ repressor sequence of the λprophage (such as A1645 and A4255); and strains deleted for the deorepressors and the deo gene (see European Patent Application PublicationNo. 0303972, published Feb. 22, 1989). Escherichia coli strain A4255(F⁺) has been deposited under ATCC Accession No. 53468, and Escherichiacoli strain A1645 has been deposited under ATCC Accession No. 67829.

The invention provides a bacterial cell which comprises these expressionplasmids. In one embodiment, the bacterial cell is an Escherichia colicell. In preferred embodiments, the invention provides an Escherichiacoli cell containing the plasmid designated pvWF-VA1, deposited in E.coli strain Sφ930 with the ATCC under ATCC Accession No. 68530;pvWF-VA3; pvWF-VB3; pvWF-VC3, deposited in E. coli strain Sφ930 with theATCC under ATCC Accession No. 68241; pvWF-VD3; or pvWF-VCL, deposited inE. coli strain 4300(F⁻) with the ATCC under ATCC Accession No. 68242.

All the E. coli host strains described above can be "cured" of theplasmids they harbor by methods well-known in the art, e.g. the ethidiumbromide method described by R. P. Novick in Bacteriol. Review 33, 210(1969).

In addition, the subject invention provides a method of producing any ofthe above-described polypeptides which comprises transforming abacterial cell with an expression plasmid encoding the polypeptide,culturing the resulting bacterial cell so that the cell produces thepolypeptide encoded by the plasmid, and recovering the polypeptide soproduced.

Furthermore, the invention provides a method of treating a subject witha cerebrovascular disorder which comprises administering to the subjectan amount of any of the polypeptides of the invention effective toinhibit platelet aggregation.

Also provided is a method of treating a subject with a cardiovasculardisorder which comprises administering to the subject an amount of apolypeptide effective to inhibit platelet aggregation. Examples ofcardiovascular disorders susceptible to treatment include acutemyocardial infarction or angina.

Further, the subject invention provides method of inhibiting plateletaggregation in a subject prior to, during, or after the subject hasundergone angioplasty, thrombolytic treatment, or coronary bypasssurgery which comprises administering to the subject an amount of apolypeptide of the invention effective to inhibit platelet aggregation.

The invention also provides a method of maintaining blood vessel patencyin a subject prior to, during, or after the subject has undergonecoronary bypass surgery, which comprises administering to the subject anamount of a polypeptide of the invention effective to inhibit plateletaggregation.

The invention also provides a method of treating a subject having cancerwhich comprises administering to the subject an amount of a polypeptideof the invention effective to retard tumor metastasis.

The invention also provides a method of inhibiting thrombosis in asubject which comprises administering to the subject an amount of apolypeptide of the invention effective to inhibit the thrombosis. Thethrombosis may be associated with an inflammatory response.

In addition, the subject invention provides a polypeptide of theinvention bound to a solid matrix.

The invention also provides a method of treating a subject sufferingfrom platelet adhesion to damaged vascular surfaces which comprisesadministering to the subject an amount of the polypeptide of theinvention effective to inhibit platelet adhesion to damaged vascularsurfaces. The range of the amount effective to inhibit platelet adhesionis 0.1-200 mg/kg body weight, preferably 1-20 mg/kg body weight. Theamount effective to inhibit platelet adhesion is the amount sufficientto maintain a blood concentration of 0.1-10 μM polypeptide. In apreferred embodiment, the blood concentration is maintained at about 1μM polypeptide.

The invention also provides a method of preventing platelet adhesion toa prosthetic material or device in a subject which comprisesadministering to the subject an amount of the polypeptide of theinvention effective to prevent platelet adhesion to the material ordevice.

The invention also provides a method of inhibiting re-occlusion in asubject following angioplasty or thrombolysis which comprisesadministering to the subject an amount of the polypeptide of theinvention effective to inhibit re-occlusion.

The invention also provides a method of preventing vaso-occlusive crisesin a subject suffering from sickle cell anemia which comprisesadministering to the subject an amount of the polypeptide of theinvention effective to prevent vaso-occlusive crises.

The invention also provides a method of preventing arteriosclerosis in asubject which comprises administering to the subject an amount of thepolypeptide of the invention effective to prevent arteriosclerosis.

The invention also provides a method of thrombolytic treatment ofthrombi-containing, platelet-rich aggregates in a subject whichcomprises administering to the subject an amount of the polypeptide ofthe invention effective to treat thrombi-containing, platelet-richaggregates.

The invention also provides a method of preventing platelet activationand thrombus formation due to high shear forces in a subject sufferingfrom stenosed or partially obstructed arteries which comprisesadministering to the subject an amount of the polypeptide of theinvention effective to prevent platelet activation and thrombusformation.

The invention also provides a method of preventing thrombin-inducedplatelet activation in a subject which comprises administering to thesubject an amount of the polypeptide of the invention effective toprevent thrombin-induced platelet activation.

The invention also provides a method of preventing stenosis as a resultof smooth muscle proliferation following vascular injury in a subjectwhich comprises administering to the subject an amount of thepolypeptide of the invention effective to prevent stenosis.

The invention also provides a method for recovering a purified,biologically active polypeptide of the invention which comprises:

(a) producing in a bacterial cell a first polypeptide having the aminoacid sequence of the polypeptide but lacking the disulfide bond;

(b) disrupting the bacterial cell so as to produce a lysate containingthe first polypeptide;

(c) treating the lysate so as to obtain inclusion bodies containing thefirst polypeptide;

(d) contacting the inclusion bodies from step (c) so as to obtain thefirst polypeptide in soluble form;

(e) treating the resulting first polypeptide so as to form thebiologically active polypeptide;

(f) recovering the biologically active polypeptide so formed; and

(g) purifying the biologically active polypeptide so recovered.

Step (e) may comprise contacting the polypeptide with a thiol-containingcompound and disulfide so as to refold and reoxidize the polypeptide.Preferably, the thiol-containing compound is glutathione, thioredoxin,β-mercaptoethanol or cysteine.

The contacting of step (d) may be effected in the presence of adenaturant such as guanidine hydrochloride or urea.

The recovery of the polypeptide in step (f) may comprise removing thedenaturant by dialysis.

In step (g), the biologically active polypeptide may be purified bycation exchange chromatography.

The first polypeptide may also be purified by cation exchangechromatography after step (d).

The invention also provides a method for treating a subject sufferingfrom von Willebrand Disease type IIb which comprises administering tothe subject an amount of the polypeptide of the invention effective toprevent binding of platelets to plasma vWF. Plasma vWF does not normallybind platelet GPIb in the circulation. GPIb binds to vWF only in thepresence of an inducer such as ristocetin or botrocetin, or if the vWFis immobilized. Patients suffering from von Willebrand Disease type IIb(vWDIIb) have a mutant vWF which spontaneously binds platelet GPIb inthe circulation, causing thrombocytopenia and a resultant bleedingdisorder due to the lack of platelets.

The examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be so construed as to,limit its scope in any way. The examples do not include detaileddescriptions for conventional methods employed in the construction ofvectors, the insertion of genes encoding polypeptides of interest intosuch vectors or the introduction of the resulting plasmids intobacterial hosts. Such methods are well-known to those skilled in the artand are described in numerous publications including Sambrook, Fritschand Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory Press, USA, (1989).

EXAMPLES

All the references to map positions correspond to the identicallynumbered positions along the translated nucleotide sequence of maturehuman von Willebrand Factor shown in FIG. 12 SEQ ID NO.1.

Example 1 Cloning and Expression of vWF GP1b Binding Domain Polypeptides

cDNA Cloning of Human vWF GP1b Binding Domain

A human endothelial cDNA library (obtained from CLONTECH Laboratories,Inc.) in λgt11 was screened for human vWF positive sequences using twosynthetic DNA probes. The probes were synthesized according to thepublished DNA sequence (Sadler et al., Proc. Nat. Acad. Sci. 82: 6394-8(1985) and Verweij et al., EMBO J. 3: 1829-47 (1986)) of human vWF(flanking 5' end and 3' end of the vWF domain known to bind the GPIbreceptor) (see FIG. 12).

The synthetic probes have the following sequences: ##STR4##

A series of vWF cDNA clones covering the entire GPIb binding domain wereidentified and isolated. The cDNA fragments were subcloned into EcoRIsite of pUC-19 (New England Biolabs, Inc.). One of the subclones,designated pvW1P (FIG. 1), contains a 2.5 Kb insert. This 2.5 Kb insertcovers the entire GPIb binding domain extending from 550 bp upstream ofthe GPIb binding site to 1100 bp downstream of the GPIb binding site.(The subclone pvW2P has also been designated pvWF-1P).

Manipulation of DNA Coding for the vWF GPIb Binding Domain

In order to obtain expression of the GPIb binding domain in Escherichiacoli under the regulation of the deo P₁ P₂ promoter, the cDNA fragmentof vWF, derived from plasmid pvW1P was used for further manipulations asdescribed below. As indicated previously, the vWF tryptic digestfragment that binds the GPIb receptor is from amino acid Val 449 toamino acid Lys 728.

A. Subcloning of the 5' end of vWF GPIb binding domain and addition of atranslation initiation codon ATG.

Plasmid pvW1P has two convenient restriction sites at the 5' end. Bsu36Iwhich cuts at the DNA sequence corresponding to amino acid Ser (445),and Tth111I which cuts at amino acid Asp (514). Synthetic fragments ofvarious size were designed that insert an ATG translation initiationcodon at the 5' end as well as additional amino acids. This was donefirst, in order to maximize the chances of obtaining high levels ofexpression. Second, they are a first step towards reducing the size ofthe vWF GPIb binding domain peptide down to the minimal size needed,possibly eliminating collagen and heparin binding sites which mayultimately interfere with the function of the product.

A1. Amino acid Glu 437 at 5' end

Synthetic oligomers with the sequences: ##STR5## were ligated to plasmidpvWF-1P digested with NdeI and Bsu36I (see FIG. 2). The plasmid obtainedwas designated pvWF-VA1. Plasmid pvWF-VA1 has been maintained in E. colistrain Sφ930 and was deposited under ATCC Accession No. 68530.

A2. Amino acid Phe 443 at 5' end

Synthetic oligomers with the sequences: ##STR6## were ligated to plasmidpvW1P digested with NdeI and Bsu36I (see FIG. 3). The plasmid obtainedwas designated pvWF-VB1.

B. Subcloning of the 3' end of vWF GPIb binding domain, introduction oftranslation stop codon.

B1. Introduction of stop codon in plasmid pVWF-VA1

A synthetic oligomer with the sequence: ##STR7## was ligated to an XmaIand HindIII digested plasmid pvWF-VA1 (see FIG. 4). The plasmid obtainedwas designated pvWF-VA2. This newly constructed plasmid contains atranslation termination codon TAA adjacent to amino acid 728 (Lys) andEcoRV site.

B2. Introduction of translation stop codon in plasmid pvWF-VB1

A synthetic oligomer with the sequence: ##STR8## was ligated to plasmidpvWF-VB1 digested with XmaI and HindIII. The plasmid obtained wasdesignated pvWF-VB2 (see FIG. 5).

Expression of the vWF GPIb binding domain in Escherichia coli

In order to obtain expression of the vWF GPIb binding domain variousexpression plasmids were constructed based on a deo P₁ P₂ constitutivepromoter system.

1. Expression of a vWF GPIb binding domain polypeptide including aminoacid Glu 437 to amino acid Lys 728 (based on plasmid pvWF-VA2)

An NdeI-EcoRV fragment was isolated from plasmid pvWF-VA2 and ligatedinto plasmid pMF-945 (see FIG. 11) digested with NdeI and PvuII (seeFIG. 6). The plasmid obtained was designated as pvWF-VA3 and wasmaintained in Escherichia coli strain Sφ930.

2. Expression of a vWF GPIb binding domain polypeptide including aminoacid Phe 443 to amino acid Lys 728 (based on plasmid pvWF-VB2)

An NdeI-EcoRV fragment was isolated from plasmid pvWF-VB2 and ligatedinto plasmid pMF-945 digested with NdeI and PvuII (see FIG. 7). Theplasmid obtained was designated as pvWF-VB3 and was maintained inEscherichia coli strain Sφ930.

3. Expression of a vWF GPIb binding domain polypeptide including aminoacid Leu 504 to amino acid Lys 728 (based on expression plasmidpvWF-VA3)

A synthetic oligomer with the sequence: ##STR9## was ligated to plasmidpvWF-VA3 digested with NdeI and Tth111I. The plasmid obtained wasdesignated as pvWF-VC3 (see FIG. 8). Plasmid pvWF-VC3 is maintained inEscherichia coli strain Sφ930 and has been deposited with the ATCC underAccession No. 68241 (also see FIG. 13).

4. Expression of a vWF GPIb binding domain polypeptide including aminoacid Leu 513 to amino acid Lys 728 (based on expression plasmidpvWF-VA3)

A synthetic oligomer with the sequence: ##STR10## was ligated to plasmidpvWF-VA3 digested with NdeI and Tth111I. The plasmid obtained wasdesignated as pVWF-VC3 (see FIG. 9). Plasmid pvWF-VD3 is maintained inEscherichia coli strain Sφ930.

Expression of vWF-GPIb binding domain polypeptides

The relative alignment of the expression plasmids is shown in FIG. 10.Plasmids pvWF-VA3, pvWF-VB3, pvWF-VC3 and pvWF-VD3 in Escherichia colistrain Sφ930 were used in order to analyze the levels of expression ofthe various vWF-GPIb binding domain peptides. The clones obtained weregrown in LB medium containing Amp (100 μg/ml) at 37° C. for 48 hours.

After 48 hours growth bacterial cells were harvested and centrifuged for2 minutes at 10,000 RPM. Pellets were dissolved in 1/10 volume of 50 mMTris-HCl pH=8.0. Sample buffer (containing SDS and β-mercaptoethanol)was added. Samples were boiled for 10 minutes and loaded on a 10% SDSpolyacrylamide gel. The expression of the vWF GPIb binding domainpolypeptides in clones pvWF-VA3, pvWF-VB3 and pvWF-VD3 was low relativeto the bacterial total proteins. The vWF polypeptides from these cloneswere detectable by Western blot analysis using commercially availablepolyclonal vWF antibody (Dekopatts a/s, Glostrup, Denmark). However,clones originated from Escherichia coli strain Sφ930 transformed withplasmid pvWF-VC3 expressed the vWF GPIb binding domain polypeptide(amino acid Leu 504 to amino acid Lys 728 plus methionine) at highlevels (as a major band) detectable upon Coomassie staining.

Escherichia coli strain Sφ930 harboring plasmid pvWF-VC3 was depositedwith the ATCC under Accession No. 68241. Subsequently, an inducibleplasmid was constructed which contains the same vWF coding region aspvWF-VC3, expressed under the control of the λP_(L) promoter and the deoribosomal binding site (see FIG. 14). This new plasmid, designatedpvWF-VCL, proved to be a high expressor of VCL, the vWF GPIb bindingdomain polypeptide (methionine plus amino acid Leu 504 to amino acid Lys728). This plasmid was deposited in Escherichia coli strain 4300 withthe ATCC under Accession No. 68242. Escherichia coli strain 4300,constructed from Escherichia coli strain ATCC Accession No. 12435, is awild-type, F⁻, biotin dependent strain, harboring the λ cI857temperature-sensitive repressor. (A third plasmid construct harboringthe same vWF coding region under the control of the λ promoter and thecII ribosomal binding site did not express any vWF peptide detectable byCoomassie staining.)

The NdeI-HindIII insert of pvWF-VCL can be conveniently subcloned intoother expression vectors such as commercially available pUC19 forproduction of a series of polypeptides which include the same amino acidsequence from amino acid 509 (cys) to amino acid 695 (cys) and have thesame biological activity.

Example 2 Fermentation of Bacteria Expressing vWF GPIb Binding DomainPolypeptides

During scale-up fermentations of clone pvWF-VC3 it was found that thehost tends to lose the plasmid due to instability. The loss of plasmidscaused a reduction in vWF GPIb binding domain polypeptide expression. Itwas found necessary to maintain continuous selective pressure (i.e.,continuous addition of Ampicillin) in order to maintain plasmid copynumber and to maintain the expression levels. Large scale fermentationwas carried out for 12 hours.

Fermentation was carried out in the following growth medium

    ______________________________________                                               N-Z amino AS   20    gr                                                       Yeast extract  10    gr                                                       NaC1           5     gr                                                       K.sub.2 HPO.sub.4                                                                            2.5   gr                                                       MgSO.sub.4 7H.sub.2 O                                                                        1.0   gr                                                       Anti foam      0.4   ml                                                ______________________________________                                    

Fructose (50%) was added to the growth medium at final concentration of150 ml/liter and Ampicillin (100 mg/ml) was pumped continuously into thefermentor (total of 8 ml/liter). Fermentation was carried out for 12hours at 37° C.

Purification of polypeptides

Cells were harvested after 12 hours fermentation and centrifuged. Thebacterial pellet obtained was resuspended in buffer 50 mM Tris pH=8.0,50 mM NaCl, 1 mM EDTA, 1 mM DTT (dithiothreitol), 1 mM PMSF(Phenylmethylsulfonyl fluoride) and 10% Glycerol!. After additionalcentrifugation and sonication the vWF GPIb binding domain polypeptidewas found in the pellet.

The vWF GPIb binding domain polypeptide was further purified bysolubilization of the pellet in 8M Urea containing 10 mM DTT, 25 mM TrispH=8 and 1 mM EDTA at room temperature. The solubilized pellet wasfractionated on a DEAE cellulose ion exchange column chromatography.(Elution buffer as above except 0.5 mM DTT).

The vWF GPIb binding domain polypeptide was eluted at 150 mM NaCl. Afterdilution to 50 mM NaCl (in the above buffer) the partially purifiedpeptide was loaded on a Q-Sepharose column. Elution from the Q-Sepharosecolumn was carried out at various NaCl concentrations (step elution).The vWF GPIb binding domain peptide was pooled in four peaks whicheluted at 100 mM, 200 mM, 250 mM and 500 mM NaCl. All four peaks weredialyzed against 150 mM NaCl and 50 mM Tris pH=8 for 36 hours. Duringthe dialysis the Urea concentration of the dialysis solution was reducedin a linear gradient from 6M Urea to no Urea.

Example 3 Biological Activity of vWF GPIb Binding Domain Polypeptides

Platelet Aggregation Assays

vWF preparation

Human plasma-derived vWF was purified from human outdated blood bankplasma according to J. Loscalzo and R. I. Handin, Biochemistry 23:3880-3886 (1984). The purified plasma-derived vWF was concentrated byAmicon 100,000 cut-off filter membrane, to a final concentration of 0.25mg/ml.

Asialo-vWF preparation

The purified plasma-derived human vWF was desialyated according to L.DeMarco and S. Shapiro, J. Clin. Invst. 68: 321-328 (1981) with thefollowing modifications:

1. The Neuraminidase used was from Vibrio cholera type II (Sigma).

2. The reaction mixture contained 0.2 Units enzyme/mg protein andprotease inhibitors according to the following concentrations:Benzamidine (20 mM), Leupeptin (15 μg/ml) and Aprotinin 20 (U/ml). Theasialo-vWF was used for platelet aggregation without any furtherpurification.

Platelet aggregation--Induced by Asialo-vWF

As stated above, soluble vWF does not bind to platelets via the GPIbreceptor. Asialo-vWF, obtained by neuraminidase treatment to removesialic acid residues, readily binds to platelets via GPIb. Presumably,the desialation lowers the net negative charge on the vWF, allowing itto bind to the negatively charged GPIb receptor. Asialo-vWF binding toplatelets causes activation, release of ADP, and GP IIb/IIIa mediatedaggregation. Platelet aggregation induced by asialo-vWF was carried outwith 200 μl of PRP (Platelet-rich plasma) (Fujimura Y., et al., J. Biol.Chem. 261: 381-385 (1986)) and 39 μg/ml of asialo-vWF (finalconcentration) in a Lumi aggregometer. The results of inhibition ofplatelet aggregation with VC, the vWF GPIb binding domain polypeptide,are summarized in Table I.

VC (also referred to as VCL or VC3) is a vWF GPIb binding domainpolypeptide which includes methionine plus amino acids 504-728 (see FIG.12).

vWF-Ristocetin induced platelet aggregation

Ristocetin-induced platelet aggregation in the presence of purifiedhuman intact vWF was carried out with washed human platelets accordingto Fujimura Y. et al., J. Biol. Chem. 261: 381-385 (1986).

The results of inhibition of platelet aggregation induced by ristocetinin the presence of intact vWF are summarized in Table II. Additionalresults using these assays are described in Example 5.

                  TABLE I                                                         ______________________________________                                        Inhibition of Asialo-vWF Induced Platelet Aggregation                         (In PRP) by VC, a vWF GPIb Binding Domain Polypeptide                                      VC           % Inhibition of                                     Q-Sepharose Frac-                                                                          concentration                                                                              Platelet Aggrega-                                   tion         μM        tion                                                ______________________________________                                        200 mM NaCl  6            76      64                                          250 mM NaCl  6            82      73                                          500 mM NaCl  6            89      79                                          ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Inhibition of Ristocetin Induced Platelet Aggregation                         by VC, a vWF GPIb Binding Domain Polypeptide                                                 VC          % Inhibition of                                    Q-Sepharose Frac-                                                                            concentration in                                                                          Platelet Aggre-                                    tion           μM       gation                                             ______________________________________                                        200 mM NaC1    10          76                                                                6           69                                                                3           38                                                                1           22                                                 250 mM NaC1    10          86                                                                6           67                                                                3           44                                                                1           34                                                 500 mM NaC1    10          100                                                               6           79                                                                3           68                                                                1           54                                                                0.25        38                                                 Dialysis Buffer                                                                              0           0                                                  (control)                                                                     ______________________________________                                    

Example 4

An Improved Method of Obtaining Pure, Oxidized, Folded and BiologicallyActive vWF GPIb Binding Domain Polypeptide

In Example 2, fermentation of cells harboring plasmid pvWF-VC3 wasdescribed. Subsequently, a preferred plasmid, pvWF-VCL was constructedas described in Example 1 and maintained in E. coli strain A4300. Thishost/plasmid system was fermented essentially as known in the art forvectors containing a gene expressed under control of the λP_(L)promoter, see, for example coassigned EPO Patent Publication No.173,280, published Mar. 5, 1986, Example 5, pages 73-74 (without addedbiotin, thiamine, trace elements, and ampicillin). In this improvedmethod of purification of vWF GPIb binding domain polypeptide, a cellpellet of the above fermentation of A4300/pvWF-VCL was used.

In this improved method a purer and more active polypeptide is producedthan by the method disclosed in Example 2. The general scheme of thedownstream process consists of steps A through H as follows:

A. Cell disruption and suspension of pellet

A pellet containing the vWF GPIb binding domain polypeptide is obtainedas described in Example 2, by sonication and centrifugation of a cellsuspension in 50 mM Tris-HCl pH=8, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mMPMSF, and 10% Glycerol.

The pellet containing the inclusion bodies is dissolved at about 10% w/vin a solution such that the final concentrations after dissolution are8M urea, 20 mM DTT, 20 mM HEPES pH 8, and 100 mM NaCl. The resultingsolution may be further purified by ion exchange chromatography asdescribed below. Alternatively, the inclusion bodies may be solubilizedin a buffer containing 6M guanidine hydrochloride followed by bufferexchange to urea. The inclusion bodies may also be dissolved atdifferent concentrations of urea, guanidine hydrochloride or any otherdenaturant or in the absence of denaturants, for example, at extremes ofpH.

B. Cation exchange chromatography

This step eliminates most of the contaminants and produces the vWF GPIbbinding domain polypeptide at greater than 90% purity. Any cationexchange (e.g. carboxymethyl) method may be used in this step, butCM-Sepharose fast flow (Pharmacia) chromatography is preferred. Thefunctional group may be carboxymethyl, a phospho group or sulphonicgroups such as sulphopropyl. The matrix may be based on inorganiccompounds, synthetic resins, polysaccharides, or organic polymers;possible matrices are agarose, cellulose, trisacryl, dextran, glassbeads, oxirane acrylic beads, acrylamide, agarose/polyacrylamidecopolymer (Ultrogel) or hydrophilic vinyl polymer (Fractogel). In aspecific embodiment, the polypeptide is loaded onto a CM-Sepharose FFcolumn equilibrated with 8M urea, 1 mM DTT, 20 mM HEPES pH 8, 100 mMNaCl. Pure polypeptide elutes in 8M urea, 1 mM DTT, 20 mM HEPES pH 8 and200 mM NaCl. Up to about 30 OD₂₈₀ units of solubilized inclusion bodiesmay be loaded per ml CM-Sepharose FF. At this ratio the elutedpolypeptide typically has a concentration of 4-5 OD₂₈₀ /ml.

C. Oxidation/Refolding

The polypeptide solution eluted from the cation exchange step above istreated with 6M guanidine hydrochloride (GuCl) to disrupt anyaggregates. The polypeptide is then diluted to 0.05 OD₂₈₀ /ml in 2MGuCl, pH 5-11, preferably 20 mM HEPES pH 8, 0.1 mM GSSG (glutathione,oxidized form). This mixture is allowed to stand overnight at roomtemperature. The products are analyzed by gel filtration on fast proteinliquid chromatography (FPLC) such as Superose 12 before proceeding.Analysis shows that this protein concentration reproducibly yields about30% correctly oxidized monomers, and 70% S-linked dimers and multimers,as well as reduced and incorrectly oxidized monomers. A higher proteinconcentration gives a higher absolute yield of correctly oxidizedmonomers but a lower percentage yield due to increased formation ofS-linked dimers and multimers. For example, a protein concentration of0.1 OD₂₈₀ /ml yields only 20% correctly oxidized monomers. Reducing theconcentration to 0.025 OD₂₈₀ /ml yields 35-40% correctly oxidizedmonomers but a lower absolute yield per liter oxidation. Oxidations mayalso be performed in urea instead of in GuCl, or in any other denaturantor in the absence of denaturants under appropriate buffer conditions inwhich, for example, pH, ionic strength, and hydrophobicity are varied.The preferred concentration of urea is in the range 0.5M to 10M,preferably 4M, and the preferred oxidant is GSSG in the range 0.01 mM to5 mM preferably 0.1 mM. Other oxidants such as CuCl₂ may be used oralternatively no oxidant may be added, thereby utilizing air oxidationonly. For scale-up, 4M urea is the presently preferred solution for theoxidation step.

D. Concentration

The oxidation products are concentrated, preferably to about OD₂₈₀ =1 bya tangential flow ultra-filtration system with a 30K cutoff membrane,such as a "MINITAN" or "PELLICON" system of Millipore. The filtrate isquite clear as the material is relatively clean and most of thecontaminants are large enough not to pass through the 30K membrane. Itis thus possible to reuse the filtrate for performing oxidations. Thisresults in considerable savings since large volumes of 2M GuCl are quiteexpensive. No difference in the oxidation products of oxidationsperformed in reused versus freshly prepared 2M GuCl was detectable byFPLC analysis.

E. Dialysis

It is necessary to reduce the GuCl or urea concentration to less than 10mM. This is achieved by dialysis against 20 mM HEPES pH8, 100 mM NaCl.The dialysis was performed in dialysis tubing with 2-3 changes ofbuffer, but may be alternatively performed by diafiltration against thesame buffer in a tangential flow ultrafiltration system with a 10K MWcutoff membrane.

During dialysis, as the concentration of GuCl (or urea or otherdenaturant) decreases, a white precipitate forms. This precipitatecontains about 80% of the protein yielded by step D comprising S--Slinked dimers, reduced and incorrectly oxidized monomer and somecontaminants which coeluted from the cation exchange step. Thesupernatant is nearly 100% correctly oxidized and refolded monomer at aconcentration of 0.2 OD₂₈₀ /ml, which is about 20% of the protein yieldof step D. This selective precipitation of contaminants and undesirableforms of the protein as a result of dialysis was surprising and notpredictable. The yield of correctly oxidized monomer can be greatlyincreased by recovery from the precipitate. This is done as follows: thesolution is clarified by centrifugation. The supernatant is saved, andthe pellet is treated with DTT to reduce S--S bonds and reoxidized asdescribed above. The pellet is dissolved in a minimal volume of 6M GuCl,20 ml HEPES pH 8, 150 mM NaCl, 20 mM DTT. The solution was passedthrough Sephadex G25 in a buffer similar to the dissolution buffer butcontaining only 1 mM DTT (instead of 20 mM). The eluate is then dilutedto OD₂₈₀ =0.05 and treated as in steps C, D and E above. This proceduremay be repeated more than once as long as additional purified monomer isobtained. All of the supernatants are then combined.

F. Cation exchange

The combined supernatant of the dialysate of step E is concentrated bybinding to CM Sepharose in 20 mM HEPES pH8, 100 mM NaCl. Elution is with20 mM HEPES pH8, 400 mM NaCl. The eluate is exclusively monomericdespite the high salt concentration. Concentrations of up to 3 mg/mlhave been achieved by this method and that is not the upper limit. Thisstep can alternatively be performed with Heparin-Sepharose which alsobinds the purified monomer in 10 mM Tris pH 7.4, 150 mM NaCl. Elutionfrom Heparin-Sepharose is performed using 10 mM Tris-HCl pH 7.4, 500 mMNaCl.

G. Dialysis

The product of the previous step is dialyzed against 20 mM HEPES pH8,150 mM NaCl.

H. Storage

At this stage the purified vWF GPIb binding domain polypeptide may belyophilized. Upon reconstitution in a volume of water equal to thevolume before lyophilization, the resultant solution containsexclusively monomeric protein showing no traces of dimers or othermultimers on FPLC.

In a specific embodiment of this method the following procedure wasperformed:

a) 10 gm inclusion bodies (comprising 0.43 g net dry weight) weredissolved in a final volume of 100 ml 8M urea, 20 mM DTT, 20 mM HEPES pH8, 100 mM NaCl.

b) The protein was loaded onto a CM Sepharose column equilibrated with8M urea, 1 mM DTT, 20 mM HEPES pH 8, 100 mM NaCl. The protein eluted at200 mM NaCL in 8M urea, 20 mM HEPES pH 8, 1 mM DTT, and was saved.

c) The saved eluate of the previous step was treated with 6M GuCl toeliminate any aggregates, and was then diluted to 0.05 OD₂₈₀ /ml in 2MGuCl, 20 mM HEPES pH 8, 0.1 mM GSSG. Oxidation was performed overnightat room temperature. (Note that the oxidation step can be performed inthe presence of urea instead of GuCl.)

d) The oxidation products were concentrated to OD₂₈₀ =1 byultrafiltration on a "MINITAN" unit containing a 30K membrane.

e) The concentrate of the previous step was dialyzed with three bufferchanges against 20 mM HEPES pH 8, 100 mM NaCl. During dialysis, as theGuCl concentration decreased, a white precipitate formed which wasremoved by centrifugation and reprocessed once as described above. Thesupernatants were combined.

f) The combined supernatants were concentrated by binding to CMSepharose in 20 mM HEPES pH 8, 100 mM NaCl. The polypeptide was elutedin 20 mM HEPES, pH 8, 400 mM NaCl and stored at 4° C.

g) The saved eluate from the previous step was dialyzed against 20 mMHEPES pH 8, 150 mM NaCl at 4° C.

h) After dialysis, the purified vWF GPIb binding domain polypeptide,designated VCL, was lyophilized.

Analysis of VCL

1. Amino acid sequence analysis of VCL purified as described aboverevealed that the N-terminal sequence is Met-Leu-His-Asp-Phe which isthe expected sequence according to FIG. 12 with the addition of anN-terminal methionine residue.

2. Examination of VCL on polyacrylamide gels revealed that VCLelectrophoreses at lower apparent molecular weight under non-reducingconditions than under reducing conditions (beta-mercaptoethanol). Thisshift from compact to less compact configuration is consistent with thereduction of a disulfide bond. Such an intramolecular bond is formedbetween the cysteines at positions 509 and 695. (The shift in molecularweight is not large enough to be consistent with the reduction of anintermolecular bond.)

Example 5

Biological Activity of VCL, a vWF GPIb binding domain polypeptide

The vwF GPIb binding domain polypeptide produced as described in Example4 was designated VCL and was assayed for biological activity asdescribed below.

1. Ristocetin induced platelet aggregation (RIPA)

RIPA assay was performed as described in Example 3 in a reaction mixcontaining 2×10⁸ platelets/ml, 1 μg/ml plasma vWF, and 1 mg/mlristocetin. A series of concentrations of VCL was tested and the IC₅₀ ofVCL in 3 assays was determined to be 0.2-0.3 μM. 100% inhibition wasachieved with about 1 μM VCL.

2. Asialo vWF induced platelet aggregation

Asialo vWF induced platelet aggregation assay was performed as describedin Example 3 with 200 μl platelet-rich plasma (PRP) and 10 μg/mlasialo-vWF in a Lumi aggregometer. A series of concentrations of VCL wastested and the IC₅₀ of VCL in this assay was determined to be 0.15 μM,and complete inhibition by 0.5 μM.

3. Effect of VCL on preformed aggregates

The effect of VCL on preformed aggregates made by RIPA was tested.Aggregates were formed as in paragraph (1) above in the absence of VCL.Addition of VCL to a concentration of 0.5 μM disrupted the aggregatesinstantaneously.

4. Inhibition of thrombin induced platelet aggregation

Thrombin induced platelet aggregation assay was performed using 0.025unit/ml thrombin and stractan prepared platelets. A series ofconcentrations of VCL was tested and the IC₅₀ of VCL in this assay wasdetermined to be 0.3 μM. This is a surprising effect, since in aparallel experiment, VCL was not effective in inhibiting direct bindingof ¹²⁵ I! labelled thrombin to platelets.

5. Effect on platelet deposition under conditions of flow

In a model system consisting of an everted denuded human umbilicalartery in a flow cell, platelet deposition may be determined. Wholehuman blood flows over the artery fragment. After 10-15 minutes, theflow is stopped and platelet deposition is determined microscopically.The IC₅₀ of VCL in this system was determined to be about 1 μM.

All the above results are summarized in Table III.

The inhibitory activity of VCL on ristocetin-induced or asialovWF-induced platelet aggregation, ristocetin-induced vWF binding, andplatelet adhesion was lost upon reduction of the disulfide bond betweenthe cysteines at positions 509 and 695. In some experiments, the reducedVCL precipitated out of solution.

                  TABLE III                                                       ______________________________________                                        Biological Activity of VCL,                                                   a vWF GPIb Binding Domain Polypeptide.                                        Example 5                                                                     (Paragraph No.)                                                                            Assay         μM VCL                                          ______________________________________                                        1            Ristocetin induced                                                                          IC.sub.50 = 0.2- 0.3                                            platelet aggregation                                             2            Asialo vWF induced                                                                          IC.sub.50 = 0.15                                                platelet aggregation                                             3            Thrombin induced                                                                            IC.sub.50 = 0.3                                                 platelet aggregation                                             4            Dissolution of                                                                              0.5                                                             preformed aggregates                                             5            Platelet deposition                                                                         IC.sub.50 = 1                                                   under conditions of                                                           flow                                                             ______________________________________                                    

Example 6 Construction of Plasmid pvWF-VEL

It was decided to construct a plasmid which expresses a slightly longerportion of the vWF GPIb binding domain than pvWF-VCL. The constructionis shown in FIGS. 15-18 and described in the brief descriptions of thefigures.

A. Construction of pvWF-VE2

Plasmid pvWF-VA2 (constructed as shown in FIG. 4) was digested with NdeIand PstI and the large fragment isolated. Four synthetic oligomers shownin FIG. 16 were prepared. Nos. 2 and 3 were treated with T4polynucleotide kinase to add 5' phosphate. The above mentioned largefragment of pvWF-VA2 was ligated as shown in FIG. 15 with the fouroligomers (two kinased, and two non-kinased). The resulting plasmidshown in FIG. 15 was designated pvWF-VE2.

B. Construction of plasmid pvWF-VE3

Plasmid pvWF-VE2 was digested with NdeI and HindIII and the 770 bpfragment containing the vWF GPIb binding domain was isolated. PlasmidpMLK-7891 was also digested with NdeI and HindIII and the large fragmentwas isolated. The resulting plasmid, shown in FIG. 17, was designatedpvWF-VE3.

C. Construction of plasmid pvWF-VEL

Plasmid pvWF-VE3 was digested with XmnI, dephosphorylated with bacterialalkaline phosphatase (BAP) and then digested with NdeI and HindIII.Plasmid pMLK-100 was digested with NdeI and HindIII and dephosphorylatedwith BAP. The two digests were then ligated to yield plasmid pvWF-VEL asshown in FIG. 18. This plasmid expresses the DNA sequence correspondingto amino acids 469-728 of mature vWF under the control of the λP_(L)promoter and the cII ribosomal binding site. The protein probably alsoincludes an additional N-terminal methionine residue. A conservativebase change was introduced into ala-473 changing GCC to GCA which alsoencodes alanine. This introduced an SphI site into the gene by changingGCCTGC to GCATGC.

Expression of pvWF-VEL in E. coli 4300(F⁻) yields a 29 kD protein whichreacts strongly with a monoclonal anti-vWF antibody and will be referredto herein as VEL.

Example 7

Pharmaceutical Uses of vWF GPIb Binding Domain Polypeptide

Examples 1 and 4 describe the production and purification of a novel vWFGPIb binding domain polypeptide designated VCL. Some of the usesenvisaged for VCL or for other vWF GPIb binding domain polypeptides aredescribed below. Pharmaceutical compositions containing VCL or suchother polypeptides may be formulated with a suitable pharmaceuticallyacceptable carrier using methods and carriers well known in the art.

1. The VCL composition described above may be used for prevention ofplatelet adhesion to damaged vascular surfaces (see Example 5,sub-section 5).

2. The VCL composition described above may be used for disruption ofplatelet-rich aggregates (see Example 5, subsection 3).

3. The VCL composition described above may be used for prevention ofre-occlusion following angioplasty or thrombolysis (see Bellinger etal., PNAS, USA, 84: 8100-8104 (1987), Prevention of occlusive coronaryartery thrombosis by a murine monoclonal antibody to porcine vonWillebrand Factor).

4. The VCL composition described above may be used for prevention ofplatelet activation and thrombus formation due to high shear forces suchas in stenosed or partially obstructed arteries or at arterialbifurcations (see Peterson et al., Blood 2: 625-628 (1987),Shear-induced platelet aggregation requires von Willebrand Factor andplatelet membrane glycoproteins Ib and IIb-IIIa).

5. The VCL composition described above may be used for prevention ofthrombosis and re-occlusion after angioplasty or thrombolysis due tothrombin activation of platelets (see Fuster et al., J. Am. Coll.Cardiol. 12: 78A-84A (1988), Antithrombotic therapy after myocardialreperfusion in acute myocardial infarction).

6. The VCL composition described above may be used for prevention ofplatelet adhesion to and aggregation on prosthetic materials (seeBadimon et al., J. of Biomaterials Applications, 5: 27-48 (1990),Platelet interaction to prosthetic materials--role of von WillebrandFactor in Platelet Interaction to PTFE).

7. The VCL composition described above may be used for prevention ofintramyocardial platelet aggregation in patients with unstable angina(see Davies et al., Circulation 73: 418-427 (1986), Intramyocardialplatelet aggregation in patients with unstable angina suffering suddenischemic cardiac death).

8. The VCL composition described above may be used for prevention ofvasospasm and vasoconstriction following arterial injury caused byangioplasty, thrombolysis or other causes (see Lam et al., Circulation75: 243-248 (1987), Is vasospasm related to platelet deposition?).

9. The VCL composition described above may be used for prevention ofrestenosis following angioplasty or thrombolysis (see McBride et al., N.Eng. J. of Med. 318: 1734-1737 (1988), Restenosis after successfulcoronary angioplasty).

10. The VCL composition described above may be used for prevention ofvaso-occlusive crises in sickle-cell anemia (see Wick et al., J. Clin.Invest. 80: 905-910 (1987), Unusually large von Willebrand multimersincrease adhesion of sickle erythrocytes to human endothelial cellsunder controlled flow).

11. The VCL composition described above may be used for prevention ofthrombosis associated with inflammatory response (see Esmon, Science235: 1348-1352 (1987), The regulation of natural anticoagulantpathways).

12. The VCL composition described above may be used for prevention ofarteriosclerosis (see Fuster et al., Circulation Res. 51: 587-593(1982), Arteriosclerosis in normal and von Willebrand pigs).

13. The VCL composition described above may be used as an antimetastaticagent (see Kitagawa et al., Cancer Res. 49: 537-541 (1989), Involvementof platelet membrane glycoprotein Ib and IIb/IIIa complex inthrombin-dependent and -independent platelet aggregations induced bytumor cells).

Example 8

1. In Vitro Studies

For these studies VCL, or vehicle control, was made up fresh in sterilewater (2.2 mg/ml stock).

A. Platelet Aggregation (PRP)

This is a standardized von Willebrand Factor (vWF)-dependent aggregationassay using human or rat platelet rich plasma (PRP). The addition ofvarious concentrations of unfractionated Bothrops jararaca venom (BJV),which includes botrocetin and an additional thrombin-like component, orristocetin results in an aggregatory response in the absence of anyadditional agent. Using ristocetin (1.5 mg/ml) as the agonist 43 μg/mlVCL abolished the aggregation of human PRP. Ristocetin up to 5.0 mg/mldid not cause measurable aggregation of rat PRP. Using this assay systemwith BJV as the agonist VCL at 83 μg/ml slightly inhibited the responseof human PRP (FIG. 19) but not rat PRP (FIG. 20).

It is concluded that ristocetin is not a suitable agonist for inducingvWF dependent aggregation in the rat. Further, it is not possible tomonitor the effects of VCL ex vivo using BJV-induced aggregation of ratPRP. However, VCL does inhibit vWF-dependent aggregation in human PRP invitro.

B. Platelet Thrombin Receptor Assay

This assay measures the inhibition of thrombin-induced plateletpro-coagulant expression and is briefly described below. Human washedplatelets are incubated in a buffer which contains CaCl₂, factor Xa,prothrombin, and human alpha-thrombin for 60 min at 28° C. At the end ofthis period an aliquot is transferred into a buffer containing S2238 andEDTA (to prevent any further thrombin generation). The S2238 reaction isterminated after 15 minutes at room temperature with acetic acid and theabsorbance at 405 nm read. The amount of S2238 cleavage directly due tothe added human alpha-thrombin is estimated by including a control whichcontains no prothrombin and this value is subtracted from all results.VCL was tested in this assay at a final concentration of 0.1 mg/ml.

This assay is sensitive to both thrombin inhibitors and thrombinreceptor antagonists. In the presence of VCL the thrombin generation was114% of control (n=2).

We therefore conclude that VCL is not a thrombin receptor antagonist inthis system.

2. In Vivo Studies

Arterial Thrombosis Model (Rat)

This method is essentially a modification of the model of Shand et al.,Thromb. Res. 45 505-515 (1987). The method we use routinely is outlinedbelow.

Rats are labelled with ¹¹¹ In platelets and ¹²⁵ I fibrinogen. The dorsalaorta is clamped, using modified Spencer-Wells forceps, for 1 minute.After a 45 minute reperfusion period the damaged vessel is removed,washed in citrate and counted. Results are expressed as mg bloodequivalents. Differences in radiolabel accumulation between placebo anddrug-treated animals are calculated and expressed as a percentageinhibition.

For the purpose of the evaluation of VCL the route of administration wasby bolus intravenous injection. VCL was used at doses of 2 mg/kg (n=5)and 4 mg/kg (n=3). It was administered 1 minute prior to clamping. Thevessel was then reperfused for twenty minutes. The antithrombotic effectwas assessed at the 20 minute end point of the reperfusion. Theshortening of the reperfusion time (as compared to routine) was designedto save compound. Appropriate vehicle controls (n=5 for both doses) wereassessed.

It can be seen that under these conditions VCL inhibits thrombusformation in this model (Table IV). The inhibition is seen for theplatelet (^(III) In) components of the thrombus at the 4 mg/kg dose. Theother changes do not reach statistical significance, thus VCL showsantithrombotic efficacy in this rat arterial model.

In conclusion, VCL exhibits an antithrombotic effect in this rat modelof arterial thrombosis which may be dose dependent.

3. Discussion

From the present data it appears that the VCL interacts with the humanplatelet vWF receptor and hence inhibits platelet aggregation in humanPRP. There is however a marked difference between species (rat vs.human) when comparing inhibition of platelet aggregation. The speciesspecificity of this effect and the causal mechanism were notinvestigated further. At a practical level this meant we were unable toanalyze ex vivo samples in order to correlate the effects of VCL onaggregation and arterial thrombosis. Hence the analysis andinterpretation of the in vivo efficacy of VCL as an antithrombotic inthe rat arterial thrombosis model is complicated by this factor.

Despite GP1b possessing binding sites for both vWF and thrombin it wouldappear that any effects of VCL on thrombin binding to GP1b do nottranslate into antagonism of thrombin-induced pro-coagulant expression.

Overall VCL shows an antithrombotic effect in the rat arterialthrombosis model. This inhibition may be due to its interference withthe binding of vWF to its receptor.

                  TABLE IV                                                        ______________________________________                                        The Effect of VCL on Arterial Thrombus                                        Formation in the Rat Dorsal Aorta                                             % INHIBITION                                                                  DOSE                                                                          (mg/kg) PLATELETS    P     FIBRINOGEN                                                                              P   N                                    ______________________________________                                        4       61.3 ± 8.0                                                                              .01   34.7 ± 8.7                                                                           NS  3                                    2        25.54 ± 20.98                                                                          NS     22.78 ± 13.48                                                                       NS  5                                    ______________________________________                                    

The results are expressed as mean percentage inhibition ± standarderror. The number of experiments in the treated groups are denoted inthe table and in all cases were compared to a group of 5 controlanimals. Statistical analysis was performed on the raw data prior totransformation to percentage inhibition. NS=not statisticallysignificant.

Example 9 Effect of VCL on In Vitro Platelet Binding to VascularComponents in a Luminar Flow Cell

The GPIb binding domain polypeptide, VCL, produced as described inExample 2 and purified as described in Example 4, was tested for itseffect on binding of platelets to different substrates naturally foundin the human vascular system. The substrates tested were endothelialcell extra-cellular matrix (ECM), fibrinogen, collagen, vWF, andfibronectin.

The test model, which has been previously described (Sakariassen et al,J. Lab. Clin. Med. 102: 522-535 (1983)), consists of a laminar flow cellinto which a glass cover slip containing the test surface is inserted.The test surface is formed by spraying or otherwise forming a layer ofthe substrate on the glass cover slip. After insertion into the flowcell, the cover slip is then subjected to perfusion with platelets underconditions of controlled flow and shear rates, and platelet adhesion isdetermined as percent coverage of the test surface by platelets.

1. ECM

Endothelial cells were grown on a glass cover slip and removed, leavinga layer of ECM as the test surface. Platelet binding was measured at twoshear rates. The results are shown in FIG. 21. Platelet coverage wasapproximately 25% and 50% at shear rates of 300 and 1300 s⁻¹respectively. The presence of 1 μM VCL in the perfusion solutioninhibited platelet adhesion to levels of about 8% and 28% coveragerespectively, which correspond to about 70% and 50% inhibitionrespectively. These figures show no significant difference in VCLinhibition at high and low shear rates.

FIG. 22 shows a dose-response curve to VCL at a shear rate of 1300 s⁻ 1from which it is possible to extrapolate a value for the IC₅₀ =0.8 μMVCL.

2. Fibrinogen

Platelet binding to a test surface of immobilized fibrinogen wasapproximately equal at the two shear rates tested (300 and 1300 s⁻¹),and in both cases was between 35-40% as shown in FIG. 23. Inhibition by1 μM VCL was approximately 40% at the lower shear rate and 75% at thehigher shear rate, representing coverage of about 22% and 10%respectively. Hantgan et al, Blood 2: 345-353, using monoclonalantibodies to α-GPIb and α-vWF (GPIb site) have shown that vWF is amajor intermediate in platelet adhesion to fibrinogen. However, to thebest of our knowledge,this is the first disclosure that a vWFpolypeptide fragment inhibits platelet adhesion to fibrin. These resultsthus demonstrate the possible use of VCL in prevention of reocclusionfollowing thrombolysis by blocking binding of platelets to thefibrin-rich residues present in blood after thrombolysis. VCL does notinhibit platelet binding to laminin (another subendothelial matrixprotein) indicating that the platelet-fibrin interaction is indeed aspecific vWF mediated reaction, while the platelet-laminin reaction isnot mediated by vWF.

3. Collagen, vWF, and Fibronectin

These three substrates were compared by preparing test surfaces of eachas described above. The results are shown in FIG. 24.

Platelet adhesion to immobilized collagen (type I) was low: about 8%coverage at 300 s⁻¹ shear rate and about 4% coverage at 1300 s⁻¹ shearrate. 1 μM VCL inhibited this adhesion by about 50%.

Platelet adhesion to test surfaces of immobilized vWF was quite high:about 20% at a shear rate of 300 s⁻¹, and about 40% at a shear rate of1300 s⁻¹. 1 μM VCL inhibited adhesion by only about 25%. This isconsistent with data demonstrating that platelet adhesion to immobilizedvWF is primarily via CPIIb-IIIA and only partially via GPIb.Interactions between platelets and immobilized vWF, particularlypurified vWF immobilized on a glass or plastic surface, are mainlythrough the GPIIb-IIIa sites rather than through the GPIb site which maybe blocked by VCL.

Platelet adhesion to immobilized fibronectin was about 10% at both 300s⁻¹ and 1300 s⁻¹ shear rates. However, VCL inhibition was only about 25%at the lower shear rate, but approximately 80% at the higher shear rate.

This is an indication that vWF mediates platelet adhesion to immobilizedfibronectin at high shear rates.

4. VCL

Platelet adhesion to immobilized VCL was also tested. Adhesion ofplatelets to VCL is shown by the data summarized in FIG. 25. Adhesion ofplatelets is ≦5% coverage at both low and high shear rates, which wasten-fold higher than adhesion to albumin under the same conditions. Thisindicates specific binding of VCL to platelets. The low coverage isprobably a characteristic of the VCL substrate being immobilized on theglass surface. Behavior in solution may be expected to differ.

The above results give an indication that VCL is particularly effectiveat inhibiting platelet adhesion to specific substrates at high shearrates. From this, it may be inferred that vWF mediates platelet bindingat high shear rates.

Since clinically relevant situations are often in regions of high shear(e.g. residual thrombus, percutaneous transluminal coronary angioplasty(PTCA)), a vWF polypeptide fragment such as the GPIb binding sitepolypeptide VCL may be of particular utility in such circumstances.

An example of the efficacy of VCL in inhibiting platelet adhesion exvivo at high shear rates is shown in Example 10.

Example 10 Ex Vivo Inhibition of Platelet Adhesion by VCL in a PorcineAorta Model

The effect of VCL was tested in a model more closely approximating invivo conditions (Badimon et al, Artiosclerosis 6: 312-320, (1986). Asection of porcine aorta was perfused in a flow cell with normal humanblood containing 90 mM sodium citrate and additionally containing ¹¹¹Indium labelled platelets. The aortic section was treated to mimic mild(MD) or severe (SD) vessel wall damage. The blood was incubated withsaline or 0.5 μM VCL for 10 minutes prior to perfusion. Followingperfusion for 5 minutes, and appropriate washing, platelet adhesion wasdetermined by measuring the tissue radioactivity.

The results, summarized in Table V, show that VCL is effective inreducing platelet deposition at the high flow rate tested, but not at alow flow rate. The effect of VCL was particularly dramatic in theexperiment mimicking severe damage where platelet deposition was reducedfrom 164±48×10⁵ /cm² to 64±15×10⁵ /cm², a reduction of approximately60%. In mildly damaged tissue, the reduction was about 50%, fromplatelet deposition of 25±7×10⁵ /cm² to 13±4×10⁵ /cm². At the low flowrate tested, VCL did not inhibit platelet deposition to seriouslydamaged tissue.

                  Table V                                                         ______________________________________                                        Effect of VCL on Platelet Adhesion to Damaaed Porcine Artery                  Ex Vivo                                                                                              Inhibitor                                              Damage type                                                                             Shear rate   Saline   VCL                                           ______________________________________                                        SD         212 S.sup.-1                                                                               55 ± 16                                                                            48 ± 18                                    SD        1690 S.sup.-1                                                                              164 ± 48                                                                            64 ± 15                                    MD        1690 S.sup.-1                                                                              25 ± 7                                                                              13 ± 4                                     ______________________________________                                    

As discussed in Example 9, since clinically relevant situations areoften in regions of high shear, a vWF GPIb binding site polypeptidefragment such as VCL may be of particular utility in such circumstances.

These results suggest that VCL is effective in reducing mural thrombusformation by inhibiting interaction between platelets and blood vesselwalls, and may be especially effective in clinical situations of highrisk of thrombosis such as stenotic disrupted atherosclerotic plaquewhich is a condition comprising severe damage at high shear rate.

Example 11 Effect of VCL in stabilizing Cardiovascular Function in an InVivo Canine coronary Artery Model

A mild stenosis was created in a canine coronary artery using acylindrical, plastic constrictor. As a result of the stenosis, thrombiform and embolize spontaneously in a cyclic manner leading to cyclicflow variations (CFV) (Ashton et al, Circulation Research 59: 568-578(1986)).

The test material is administered after establishment of CFV. The resultis expressed as the percentage of animals in which CFV is abolished as aresult of administration of the material being tested. Subsequently,renewed platelet aggregation is induced by the administration ofepinephrine. The percentage of treated animals in which epinephrineadministration induces CFV as a result of renewed platelet aggregationis a further indication of the efficacy of the materials underevaluation.

Two materials, VCL, a GPIb binding domain polypeptide, and a thromboxaneA₂ (TXA₂) receptor antagonist were tested to determine their effect inthis model. VCL was administered at 0.5 mg/kg i.v. bolus plus 0.25mg/kg/hr i.v infusion to maintain blood concentration of about 0.3 μM,based on 1 liter of blood representing approximately 14 kg of bodyweight.

The results are summarized in Table VI. VCL abolished CFV in 43% of theanimals, the TXA₂ receptor antagonist in 71% of the animals, and the twomaterials in combination abolished CFV in 100% of the animals.

Subsequent administration of epinephrine reestablished CFV in 100% ofthe animals treated with only one of the two materials tested, but inonly 50% of the animals treated with the combination of both materials.

Furthermore, as may be seen from Table VI, the dose of epinephrinerequired to restore CFV was higher in the animals treated with VCL (0.3μg/kg/min) than in animals treated with the TXA2 receptor antagonist(0.2 μg/kg/min), and at least double in the animals treated with both(0.6 μg/kg/min). Thus VCL provides greater protection againstepinephrine induced CFV than TXA₂ receptor antagonist, and use of VCL incombination with TXA₂ receptor antagonist provides at least double theprotection of either agent used alone.

                  Table VI                                                        ______________________________________                                        In Vivo Effect of VCL on CFV in Dogs                                                                          % Restoration of                                                   Epinephrine                                                                              CFV by Epinephrine                                      % Abolition                                                                              Dosage     Induced Platelet                              Test Compound                                                                           of CFV     μg/kg/min                                                                             Aggregation                                   ______________________________________                                        VCL       43         0.3        100                                           TXA.sub.2 receptor                                                                      71         0.2        100                                           antagonist                                                                    VCL +     100        0.6        50                                            TXA.sub.2 receptor                                                            antagonist                                                                    ______________________________________                                    

These results are an indication that VCL confers increased protectionagainst CFV and against epinephrine induced platelet aggregation.

Example 12 Effect of VCL on Bleeding in a Baboon

A model using a baboon for vascular studies has been described (Kelly etal, Blood 77: 1006-1012, (March 1991)). Preliminary results indicatethat administration of 10 μM VCL increased bleeding time from 4 minutesto 30 minutes without affecting platelet viability or causingthrombocytopenia. These are attributes required of an inhibitor ofplatelet adhesion to subendothelium.

Based on these results and the results presented in Example 11, probabledosages would be in range of 0.1-200 mg/kg body weight, preferably 1-20mg/kg body weight, based on 1 L blood representing about 14 kg bodyweight. It might be necessary for further i.v. administration tomaintain a blood concentration of about 1 μM.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6153 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..6153                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGCCTATCCTGTCGGCCCCCCATGGTCAAGCTGGTGTGTCCCGCTGAC48                            SerLeuSerCysArgProProMetValLysLeuValCysProAlaAsp                              151015                                                                        AACCTGCGGGCTGAAGGGCTCGAGTGTACCAAAACGTGCCAGAACTAT96                            AsnLeuArgAlaGluGlyLeuGluCysThrLysThrCysGlnAsnTyr                              202530                                                                        GACCTGGAGTGCATGAGCATGGGCTGTGTCTCTGGCTGCCTCTGCCCC144                           AspLeuGluCysMetSerMetGlyCysValSerGlyCysLeuCysPro                              354045                                                                        CCGGGCATGGTCCGGCATGAGAACAGATGTGTGGCCCTGGAAAGGTGT192                           ProGlyMetValArgHisGluAsnArgCysValAlaLeuGluArgCys                              505560                                                                        CCCTGCTTCCATCAGGGCAAGGAGTATGCCCCTGGAGAAACAGTGAAG240                           ProCysPheHisGlnGlyLysGluTyrAlaProGlyGluThrValLys                              65707580                                                                      ATTGGCTGCAACACTTGTGTCTGTCGGGACCGGAAGTGGAACTGCACA288                           IleGlyCysAsnThrCysValCysArgAspArgLysTrpAsnCysThr                              859095                                                                        GACCATGTGTGTGATGCCACGTGCTCCACGATCGGCATGGCCCACTAC336                           AspHisValCysAspAlaThrCysSerThrIleGlyMetAlaHisTyr                              100105110                                                                     CTCACCTTCGACGGGCTCAAATACCTGTTCCCCGGGGAGTGCCAGTAC384                           LeuThrPheAspGlyLeuLysTyrLeuPheProGlyGluCysGlnTyr                              115120125                                                                     GTTCTGGTGCAGGATTACTGCGGCAGTAACCCTGGGACCTTTCGGATC432                           ValLeuValGlnAspTyrCysGlySerAsnProGlyThrPheArgIle                              130135140                                                                     CTAGTGGGGAATAAGGGATGCAGCCACCCCTCAGTGAAATGCAAGAAA480                           LeuValGlyAsnLysGlyCysSerHisProSerValLysCysLysLys                              145150155160                                                                  CGGGTCACCATCCTGGTGGAGGGAGGAGAGATTGAGCTGTTTGACGGG528                           ArgValThrIleLeuValGluGlyGlyGluIleGluLeuPheAspGly                              165170175                                                                     GAGGTGAATGTGAAGAGGCCCATGAAGGATGAGACTCACTTTGAGGTG576                           GluValAsnValLysArgProMetLysAspGluThrHisPheGluVal                              180185190                                                                     GTGGAGTCTGGCCGGTACATCATTCTGCTGCTGGGCAAAGCCCTCTCC624                           ValGluSerGlyArgTyrIleIleLeuLeuLeuGlyLysAlaLeuSer                              195200205                                                                     GTGGTCTGGGACCGCCACCTGAGCATCTCCGTGGTCCTGAAGCAGACA672                           ValValTrpAspArgHisLeuSerIleSerValValLeuLysGlnThr                              210215220                                                                     TACCAGGAGAAAGTGTGTGGCCTGTGTGGGAATTTTGATGGCATCCAG720                           TyrGlnGluLysValCysGlyLeuCysGlyAsnPheAspGlyIleGln                              225230235240                                                                  AACAATGACCTCACCAGCAGCAACCTCCAAGTGGAGGAGGACCCTGTG768                           AsnAsnAspLeuThrSerSerAsnLeuGlnValGluGluAspProVal                              245250255                                                                     GACTTTGGGAAGTCCTGGGAAGTGAGCTCGCAGTGTGCTGACACCAGA816                           AspPheGlyLysSerTrpGluValSerSerGlnCysAlaAspThrArg                              260265270                                                                     AAAGTGCCTCTGGACTCATCCCCTGCCACCTGCCATAACAACATCATG864                           LysValProLeuAspSerSerProAlaThrCysHisAsnAsnIleMet                              275280285                                                                     AAGCAGACGATGGTGGATTCCTCCTGTAGAATCCTTACCAGTGACGTC912                           LysGlnThrMetValAspSerSerCysArgIleLeuThrSerAspVal                              290295300                                                                     TTCCAGGACTGCAACAAGCTGGTGGACCCCGAGCCATATCTGGATGTC960                           PheGlnAspCysAsnLysLeuValAspProGluProTyrLeuAspVal                              305310315320                                                                  TGCATTTACGACACCTGCTCCTGTGAGTCCATTGGGGACTGCGCCTGC1008                          CysIleTyrAspThrCysSerCysGluSerIleGlyAspCysAlaCys                              325330335                                                                     TTCTGCGACACCATTGCTGCCTATGCCCACGTGTGTGCCCAGCATGGC1056                          PheCysAspThrIleAlaAlaTyrAlaHisValCysAlaGlnHisGly                              340345350                                                                     AAGGTGGTGACCTGGAGGACGGCCACATTGTGCCCCCAGAGCTGCGAG1104                          LysValValThrTrpArgThrAlaThrLeuCysProGlnSerCysGlu                              355360365                                                                     GAGAGGAATCTCCGGGAGAACGGGTATGAGTGTGAGTGGCGCTATAAC1152                          GluArgAsnLeuArgGluAsnGlyTyrGluCysGluTrpArgTyrAsn                              370375380                                                                     AGCTGTGCACCTGCCTGTCAAGTCACGTGTCAGCACCCTGAGCCACTG1200                          SerCysAlaProAlaCysGlnValThrCysGlnHisProGluProLeu                              385390395400                                                                  GCCTGCCCTGTGCAGTGTGTGGAGGGCTGCCATGCCCATTGCCCTCCA1248                          AlaCysProValGlnCysValGluGlyCysHisAlaHisCysProPro                              405410415                                                                     GGCAAAATCCTGGATGAGCTTTTGCAGACCTGCGTTGACCCTGAAGAC1296                          GlyLysIleLeuAspGluLeuLeuGlnThrCysValAspProGluAsp                              420425430                                                                     TGTCCAGTGTGTGAGGTGGCTGGCCGGCGTTTTGCCTCAGGAAAGAAA1344                          CysProValCysGluValAlaGlyArgArgPheAlaSerGlyLysLys                              435440445                                                                     GTCACCTTGAATCCCAGTGACCCTGAGCACTGCCAGATTTGCCACTGT1392                          ValThrLeuAsnProSerAspProGluHisCysGlnIleCysHisCys                              450455460                                                                     GATGTTGTCAACCTCACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTG1440                          AspValValAsnLeuThrCysGluAlaCysGlnGluProGlyGlyLeu                              465470475480                                                                  GTGGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTCTGTATGTG1488                          ValValProProThrAspAlaProValSerProThrThrLeuTyrVal                              485490495                                                                     GAGGACATCTCGGAACCGCCGTTGCACGATTTCTACTGCAGCAGGCTA1536                          GluAspIleSerGluProProLeuHisAspPheTyrCysSerArgLeu                              500505510                                                                     CTGGACCTGGTCTTCCTGCTGGATGGCTCCTCCAGGCTGTCCGAGGCT1584                          LeuAspLeuValPheLeuLeuAspGlySerSerArgLeuSerGluAla                              515520525                                                                     GAGTTTGAAGTGCTGAAGGCCTTTGTGGTGGACATGATGGAGCGGCTG1632                          GluPheGluValLeuLysAlaPheValValAspMetMetGluArgLeu                              530535540                                                                     CGCATCTCCCAGAAGTGGGTCCGCGTGGCCGTGGTGGAGTACCACGAC1680                          ArgIleSerGlnLysTrpValArgValAlaValValGluTyrHisAsp                              545550555560                                                                  GGCTCCCACGCCTACATCGGGCTCAAGGACCGGAAGCGACCATCAGAG1728                          GlySerHisAlaTyrIleGlyLeuLysAspArgLysArgProSerGlu                              565570575                                                                     CTGCGGCGCATTGCCAGCCAGGTGAAGTATGCGGGCAGCCAGGTGGCC1776                          LeuArgArgIleAlaSerGlnValLysTyrAlaGlySerGlnValAla                              580585590                                                                     TCCACCAGCGAGGTCTTGAAATACACACTGTTCCAAATCTTCAGCAAG1824                          SerThrSerGluValLeuLysTyrThrLeuPheGlnIlePheSerLys                              595600605                                                                     ATCGACCGCCCTGAAGCCTCCCGCATCGCCCTGCTCCTGATGGCCAGC1872                          IleAspArgProGluAlaSerArgIleAlaLeuLeuLeuMetAlaSer                              610615620                                                                     CAGGAGCCCCAACGGATGTCCCGGAACTTTGTCCGCTACGTCCAGGGC1920                          GlnGluProGlnArgMetSerArgAsnPheValArgTyrValGlnGly                              625630635640                                                                  CTGAAGAAGAAGAAGGTCATTGTGATCCCGGTGGGCATTGGGCCCCAT1968                          LeuLysLysLysLysValIleValIleProValGlyIleGlyProHis                              645650655                                                                     GCCAACCTCAAGCAGATCCGCCTCATCGAGAAGCAGGCCCCTGAGAAC2016                          AlaAsnLeuLysGlnIleArgLeuIleGluLysGlnAlaProGluAsn                              660665670                                                                     AAGGCCTTCGTGCTGAGCAGTGTGGATGAGCTGGAGCAGCAAAGGGAC2064                          LysAlaPheValLeuSerSerValAspGluLeuGluGlnGlnArgAsp                              675680685                                                                     GAGATCGTTAGCTACCTCTGTGACCTTGCCCCTGAAGCCCCTCCTCCT2112                          GluIleValSerTyrLeuCysAspLeuAlaProGluAlaProProPro                              690695700                                                                     ACTCTGCCCCCCGACATGGCACAAGTCACTGTGGGCCCGGGGCTCTTG2160                          ThrLeuProProAspMetAlaGlnValThrValGlyProGlyLeuLeu                              705710715720                                                                  GGGGTTTCGACCCTGGGGCCCAAGAGGAACTCCATGGTTCTGGATGTG2208                          GlyValSerThrLeuGlyProLysArgAsnSerMetValLeuAspVal                              725730735                                                                     GCGTTCGTCCTGGAAGGATCGGACAAAATTGGTGAAGCCGACTTCAAC2256                          AlaPheValLeuGluGlySerAspLysIleGlyGluAlaAspPheAsn                              740745750                                                                     AGGAGCAAGGAGTTCATGGAGGAGGTGATTCAGCGGATGGATGTGGGC2304                          ArgSerLysGluPheMetGluGluValIleGlnArgMetAspValGly                              755760765                                                                     CAGGACAGCATCCACGTCACGGTGCTGCAGTACTCCTACATGGTGACC2352                          GlnAspSerIleHisValThrValLeuGlnTyrSerTyrMetValThr                              770775780                                                                     GTGGAGTACCCCTTCAGCGAGGCACAGTCCAAAGGGGACATCCTGCAG2400                          ValGluTyrProPheSerGluAlaGlnSerLysGlyAspIleLeuGln                              785790795800                                                                  CGGGTGCGAGAGATCCGCTACCAGGGCGGCAACAGGACCAACACTGGG2448                          ArgValArgGluIleArgTyrGlnGlyGlyAsnArgThrAsnThrGly                              805810815                                                                     CTGGCCCTGCGGTACCTCTCTGACCACAGCTTCTTGGTCAGCCAGGGT2496                          LeuAlaLeuArgTyrLeuSerAspHisSerPheLeuValSerGlnGly                              820825830                                                                     GACCGGGAGCAGGCGCCCAACCTGGTCTACATGGTCACCGGAAATCCT2544                          AspArgGluGlnAlaProAsnLeuValTyrMetValThrGlyAsnPro                              835840845                                                                     GCCTCTGATGAGATCAAGAGGCTGCCTGGAGACATCCAGGTGGTGCCC2592                          AlaSerAspGluIleLysArgLeuProGlyAspIleGlnValValPro                              850855860                                                                     ATTGGAGTGGGCCCTAATGCCAACGTGCAGGAGCTGGAGAGGATTGGC2640                          IleGlyValGlyProAsnAlaAsnValGlnGluLeuGluArgIleGly                              865870875880                                                                  TGGCCCAATGCCCCTATCCTCATCCAGGACTTTGAGACGCTCCCCCGA2688                          TrpProAsnAlaProIleLeuIleGlnAspPheGluThrLeuProArg                              885890895                                                                     GAGGCTCCTGACCTGGTGCTGCAGAGGTGCTGCTCCGGAGAGGGGCTG2736                          GluAlaProAspLeuValLeuGlnArgCysCysSerGlyGluGlyLeu                              900905910                                                                     CAGATCCCCACCCTCTCCCCAGCACCTGACTGCAGCCAGCCCCTGGAC2784                          GlnIleProThrLeuSerProAlaProAspCysSerGlnProLeuAsp                              915920925                                                                     GTGATCCTTCTCCTGGATGGCTCCTCCAGTTTCCCAGCTTCTTATTTT2832                          ValIleLeuLeuLeuAspGlySerSerSerPheProAlaSerTyrPhe                              930935940                                                                     GATGAAATGAAGAGTTTCGCCAAGGCTTTCATTTCAAAAGCCAATATA2880                          AspGluMetLysSerPheAlaLysAlaPheIleSerLysAlaAsnIle                              945950955960                                                                  GGGCCTCGTCTCACTCAGGTGTCAGTGCTGCAGTATGGAAGCATCACC2928                          GlyProArgLeuThrGlnValSerValLeuGlnTyrGlySerIleThr                              965970975                                                                     ACCATTGACGTGCCATGGAACGTGGTCCCGGAGAAAGCCCATTTGCTG2976                          ThrIleAspValProTrpAsnValValProGluLysAlaHisLeuLeu                              980985990                                                                     AGCCTTGTGGACGTCATGCAGCGGGAGGGAGGCCCCAGCCAAATCGGG3024                          SerLeuValAspValMetGlnArgGluGlyGlyProSerGlnIleGly                              99510001005                                                                   GATGCCTTGGGCTTTGCTGTGCGATACTTGACTTCAGAAATGCATGGT3072                          AspAlaLeuGlyPheAlaValArgTyrLeuThrSerGluMetHisGly                              101010151020                                                                  GCCAGGCCGGGAGCCTCAAAGGCGGTGGTCATCCTGGTCACGGACGTC3120                          AlaArgProGlyAlaSerLysAlaValValIleLeuValThrAspVal                              1025103010351040                                                              TCTGTGGATTCAGTGGATGCAGCAGCTGATGCCGCCAGGTCCAACAGA3168                          SerValAspSerValAspAlaAlaAlaAspAlaAlaArgSerAsnArg                              104510501055                                                                  GTGACAGTGTTCCCTATTGGAATTGGAGATCGCTACGATGCAGCCCAG3216                          ValThrValPheProIleGlyIleGlyAspArgTyrAspAlaAlaGln                              106010651070                                                                  CTACGGATCTTGGCAGGCCCAGCAGGCGACTCCAACGTGGTGAAGCTC3264                          LeuArgIleLeuAlaGlyProAlaGlyAspSerAsnValValLysLeu                              107510801085                                                                  CAGCGAATCGAAGACCTCCCTACCATGGTCACCTTGGGCAATTCCTTC3312                          GlnArgIleGluAspLeuProThrMetValThrLeuGlyAsnSerPhe                              109010951100                                                                  CTCCACAAACTGTGCTCTGGATTTGTTAGGATTTGCATGGATGAGGAT3360                          LeuHisLysLeuCysSerGlyPheValArgIleCysMetAspGluAsp                              1105111011151120                                                              GGGAATGAGAAGAGGCCCGGGGACGTCTGGACCTTGCCAGACCAGTGC3408                          GlyAsnGluLysArgProGlyAspValTrpThrLeuProAspGlnCys                              112511301135                                                                  CACACCGTGACTTGCCAGCCAGATGGCCAGACCTTGCTGAAGAGTCAT3456                          HisThrValThrCysGlnProAspGlyGlnThrLeuLeuLysSerHis                              114011451150                                                                  CGGGTCAACTGTGACCGGGGGCTGAGGCCTTCGTGCCCTAACAGCCAG3504                          ArgValAsnCysAspArgGlyLeuArgProSerCysProAsnSerGln                              115511601165                                                                  TCCCCTGTTAAAGTGGAAGAGACCTGTGGCTGCCGCTGGACCTGCCCC3552                          SerProValLysValGluGluThrCysGlyCysArgTrpThrCysPro                              117011751180                                                                  TGCGTGTGCACAGGCAGCTCCACTCGGCACATCGTGACCTTTGATGGG3600                          CysValCysThrGlySerSerThrArgHisIleValThrPheAspGly                              1185119011951200                                                              CAGAATTTCAAGCTGACTGGCAGCTGTTCTTATGTCCTATTTCAAAAC3648                          GlnAsnPheLysLeuThrGlySerCysSerTyrValLeuPheGlnAsn                              120512101215                                                                  AAGGAGCAGGACCTGGAGGTGATTCTCCATAATGGTGCCTGCAGCCCT3696                          LysGluGlnAspLeuGluValIleLeuHisAsnGlyAlaCysSerPro                              122012251230                                                                  GGAGCAAGGCAGGGCTGCATGAAATCCATCGAGGTGAAGCACAGTGCC3744                          GlyAlaArgGlnGlyCysMetLysSerIleGluValLysHisSerAla                              123512401245                                                                  CTCTCCGTCGAGCTGCACAGTGACATGGAGGTGACGGTGAATGGGAGA3792                          LeuSerValGluLeuHisSerAspMetGluValThrValAsnGlyArg                              125012551260                                                                  CTGGTCTCTGTTCCTTACGTGGGTGGGAACATGGAAGTCAACGTTTAT3840                          LeuValSerValProTyrValGlyGlyAsnMetGluValAsnValTyr                              1265127012751280                                                              GGTGCCATCATGCATGAGGTCAGATTCAATCACCTTGGTCACATCTTC3888                          GlyAlaIleMetHisGluValArgPheAsnHisLeuGlyHisIlePhe                              128512901295                                                                  ACATTCACTCCACAAAACAATGAGTTCCAACTGCAGCTCAGCCCCAAG3936                          ThrPheThrProGlnAsnAsnGluPheGlnLeuGlnLeuSerProLys                              130013051310                                                                  ACTTTTGCTTCAAAGACGTATGGTCTGTGTGGGATCTGTGATGAGAAC3984                          ThrPheAlaSerLysThrTyrGlyLeuCysGlyIleCysAspGluAsn                              131513201325                                                                  GGAGCCAATGACTTCATGCTGAGGGATGGCACAGTCACCACAGACTGG4032                          GlyAlaAsnAspPheMetLeuArgAspGlyThrValThrThrAspTrp                              133013351340                                                                  AAAACACTTGTTCAGGAATGGACTGTGCAGCGGCCAGGACAGACGTGC4080                          LysThrLeuValGlnGluTrpThrValGlnArgProGlyGlnThrCys                              1345135013551360                                                              CAGCCCATCCTGGAGGAGCAGTGTCTTGTCCCCGACAGCTCCCACTGC4128                          GlnProIleLeuGluGluGlnCysLeuValProAspSerSerHisCys                              136513701375                                                                  CAGGTCCTCCTCTTACCACTGTTTGCTGAATGCCACAAGGTCCTGGCT4176                          GlnValLeuLeuLeuProLeuPheAlaGluCysHisLysValLeuAla                              138013851390                                                                  CCAGCCACATTCTATGCCATCTGCCAGCAGGACAGTTCGCACCAGGAG4224                          ProAlaThrPheTyrAlaIleCysGlnGlnAspSerSerHisGlnGlu                              139514001405                                                                  CAAGTGTGTGAGGTGATCGCCTCTTATGCCCACCTCTGTCGGACCAAC4272                          GlnValCysGluValIleAlaSerTyrAlaHisLeuCysArgThrAsn                              141014151420                                                                  GGGGTCTGCGTTGACTGGAGGACACCTGATTTCTGTGCTATGTCATGC4320                          GlyValCysValAspTrpArgThrProAspPheCysAlaMetSerCys                              1425143014351440                                                              CCACCATCTCTGGTCTACAACCACTGTGAGCATGGCTGTCCCCGGCAC4368                          ProProSerLeuValTyrAsnHisCysGluHisGlyCysProArgHis                              144514501455                                                                  TGTGATGGCAACGTGAGCTCCTGTGGGGACCATCCCTCCGAAGGCTGT4416                          CysAspGlyAsnValSerSerCysGlyAspHisProSerGluGlyCys                              146014651470                                                                  TTCTGCCCTCCAGATAAAGTCATGTTGGAAGGCAGCTGTGTCCCTGAA4464                          PheCysProProAspLysValMetLeuGluGlySerCysValProGlu                              147514801485                                                                  GAGGCCTGCACTCAGTGCATTGGTGAGGATGGAGTCCAGCACCAGTTC4512                          GluAlaCysThrGlnCysIleGlyGluAspGlyValGlnHisGlnPhe                              149014951500                                                                  CTGGAAGCCTGGGTCCCGGACCACCAGCCCTGTCAGATCTGCACATGC4560                          LeuGluAlaTrpValProAspHisGlnProCysGlnIleCysThrCys                              1505151015151520                                                              CTCAGCGGGCGGAAGGTCAACTGCACAACGCAGCCCTGCCCCACGGCC4608                          LeuSerGlyArgLysValAsnCysThrThrGlnProCysProThrAla                              152515301535                                                                  AAAGCTCCCACGTGTGGCCTGTGTGAAGTAGCCCGCCTCCGCCAGAAT4656                          LysAlaProThrCysGlyLeuCysGluValAlaArgLeuArgGlnAsn                              154015451550                                                                  GCAGACCAGTGCTGCCCCGAGTATGAGTGTGTGTGTGACCCAGTGAGC4704                          AlaAspGlnCysCysProGluTyrGluCysValCysAspProValSer                              155515601565                                                                  TGTGACCTGCCCCCAGTGCCTCACTGTGAACGTGGCCTCCAGCCCACA4752                          CysAspLeuProProValProHisCysGluArgGlyLeuGlnProThr                              157015751580                                                                  CTGACCAACCCTGGCGAGTGCAGACCCAACTTCACCTGCGCCTGCAGG4800                          LeuThrAsnProGlyGluCysArgProAsnPheThrCysAlaCysArg                              1585159015951600                                                              AAGGAGGAGTGCAAAAGAGTGTCCCCACCCTCCTGCCCCCCGCACCGT4848                          LysGluGluCysLysArgValSerProProSerCysProProHisArg                              160516101615                                                                  TTGCCCACCCTTCGGAAGACCCAGTGCTGTGATGAGTATGAGTGTGCC4896                          LeuProThrLeuArgLysThrGlnCysCysAspGluTyrGluCysAla                              162016251630                                                                  TGCAACTGTGTCAACTCCACAGTGAGCTGTCCCCTTGGGTACTTGGCC4944                          CysAsnCysValAsnSerThrValSerCysProLeuGlyTyrLeuAla                              163516401645                                                                  TCAACCGCCACCAATGACTGTGGCTGTACCACAACCACCTGCCTTCCC4992                          SerThrAlaThrAsnAspCysGlyCysThrThrThrThrCysLeuPro                              165016551660                                                                  GACAAGGTGTGTGTCCACCGAAGCACCATCTACCCTGTGGGCCAGTTC5040                          AspLysValCysValHisArgSerThrIleTyrProValGlyGlnPhe                              1665167016751680                                                              TGGGAGGAGGGCTGCGATGTGTGCACCTGCACCGACATGGAGGATGCC5088                          TrpGluGluGlyCysAspValCysThrCysThrAspMetGluAspAla                              168516901695                                                                  GTGATGGGCCTCCGCGTGGCCCAGTGCTCCCAGAAGCCCTGTGAGGAC5136                          ValMetGlyLeuArgValAlaGlnCysSerGlnLysProCysGluAsp                              170017051710                                                                  AGCTGTCGGTCGGGCTTCACTTACGTTCTGCATGAAGGCGAGTGCTGT5184                          SerCysArgSerGlyPheThrTyrValLeuHisGluGlyGluCysCys                              171517201725                                                                  GGAAGGTGCCTGCCATCTGCCTGTGAGGTGGTGACTGGCTCACCGCGG5232                          GlyArgCysLeuProSerAlaCysGluValValThrGlySerProArg                              173017351740                                                                  GGGGACTCCCAGTCTTCCTGGAAGAGTGTCGGCTCCCAGTGGGCCTCC5280                          GlyAspSerGlnSerSerTrpLysSerValGlySerGlnTrpAlaSer                              1745175017551760                                                              CCGGAGAACCCCTGCCTCATCAATGAGTGTGTCCGAGTGAAGGAGGAG5328                          ProGluAsnProCysLeuIleAsnGluCysValArgValLysGluGlu                              176517701775                                                                  GTCTTTATACAACAAAGGAACGTCTCCTGCCCCCAGCTGGAGGTCCCT5376                          ValPheIleGlnGlnArgAsnValSerCysProGlnLeuGluValPro                              178017851790                                                                  GTCTGCCCCTCGGGCTTTCAGCTGAGCTGTAAGACCTCAGCGTGCTGC5424                          ValCysProSerGlyPheGlnLeuSerCysLysThrSerAlaCysCys                              179518001805                                                                  CCAAGCTGTCGCTGTGAGCGCATGGAGGCCTGCATGCTCAATGGCACT5472                          ProSerCysArgCysGluArgMetGluAlaCysMetLeuAsnGlyThr                              181018151820                                                                  GTCATTGGGCCCGGGAAGACTGTGATGATCGATGTGTGCACGACCTGC5520                          ValIleGlyProGlyLysThrValMetIleAspValCysThrThrCys                              1825183018351840                                                              CGCTGCATGGTGCAGGTGGGGGTCATCTCTGGATTCAAGCTGGAGTGC5568                          ArgCysMetValGlnValGlyValIleSerGlyPheLysLeuGluCys                              184518501855                                                                  AGGAAGACCACCTGCAACCCCTGCCCCCTGGGTTACAAGGAAGAAAAT5616                          ArgLysThrThrCysAsnProCysProLeuGlyTyrLysGluGluAsn                              186018651870                                                                  AACACAGGTGAATGTTGTGGGAGATGTTTGCCTACGGCTTGCACCATT5664                          AsnThrGlyGluCysCysGlyArgCysLeuProThrAlaCysThrIle                              187518801885                                                                  CAGCTAAGAGGAGGACAGATCATGACACTGAAGCGTGATGAGACGCTC5712                          GlnLeuArgGlyGlyGlnIleMetThrLeuLysArgAspGluThrLeu                              189018951900                                                                  CAGGATGGCTGTGATACTCACTTCTGCAAGGTCAATGAGAGAGGAGAG5760                          GlnAspGlyCysAspThrHisPheCysLysValAsnGluArgGlyGlu                              1905191019151920                                                              TACTTCTGGGAGAAGAGGGTCACAGGCTGCCCACCCTTTGATGAACAC5808                          TyrPheTrpGluLysArgValThrGlyCysProProPheAspGluHis                              192519301935                                                                  AAGTGTCTGGCTGAGGGAGGTAAAATTATGAAAATTCCAGGCACCTGC5856                          LysCysLeuAlaGluGlyGlyLysIleMetLysIleProGlyThrCys                              194019451950                                                                  TGTGACACATGTGAGGAGCCTGAGTGCAACGACATCACTGCCAGGCTG5904                          CysAspThrCysGluGluProGluCysAsnAspIleThrAlaArgLeu                              195519601965                                                                  CAGTATGTCAAGGTGGGAAGCTGTAAGTCTGAAGTAGAGGTGGATATC5952                          GlnTyrValLysValGlySerCysLysSerGluValGluValAspIle                              197019751980                                                                  CACTACTGCCAGGGCAAATGTGCCAGCAAAGCCATGTACTCCATTGAC6000                          HisTyrCysGlnGlyLysCysAlaSerLysAlaMetTyrSerIleAsp                              1985199019952000                                                              ATCAACGATGTGCAGGACCAGTGCTCCTGCTGCTCTCCGACACGGACG6048                          IleAsnAspValGlnAspGlnCysSerCysCysSerProThrArgThr                              200520102015                                                                  GAGCCCATGCAGGTGGCCCTGCACTGCACCAATGGCTCTGTTGTGTAC6096                          GluProMetGlnValAlaLeuHisCysThrAsnGlySerValValTyr                              202020252030                                                                  CATGAGGTTCTCAATGCCATGGAGTGCAAATGCTCCCCCAGGAAGTGC6144                          HisGluValLeuAsnAlaMetGluCysLysCysSerProArgLysCys                              203520402045                                                                  AGCAAGTGA6153                                                                 SerLys                                                                        2050                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2050 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerLeuSerCysArgProProMetValLysLeuValCysProAlaAsp                              151015                                                                        AsnLeuArgAlaGluGlyLeuGluCysThrLysThrCysGlnAsnTyr                              202530                                                                        AspLeuGluCysMetSerMetGlyCysValSerGlyCysLeuCysPro                              354045                                                                        ProGlyMetValArgHisGluAsnArgCysValAlaLeuGluArgCys                              505560                                                                        ProCysPheHisGlnGlyLysGluTyrAlaProGlyGluThrValLys                              65707580                                                                      IleGlyCysAsnThrCysValCysArgAspArgLysTrpAsnCysThr                              859095                                                                        AspHisValCysAspAlaThrCysSerThrIleGlyMetAlaHisTyr                              100105110                                                                     LeuThrPheAspGlyLeuLysTyrLeuPheProGlyGluCysGlnTyr                              115120125                                                                     ValLeuValGlnAspTyrCysGlySerAsnProGlyThrPheArgIle                              130135140                                                                     LeuValGlyAsnLysGlyCysSerHisProSerValLysCysLysLys                              145150155160                                                                  ArgValThrIleLeuValGluGlyGlyGluIleGluLeuPheAspGly                              165170175                                                                     GluValAsnValLysArgProMetLysAspGluThrHisPheGluVal                              180185190                                                                     ValGluSerGlyArgTyrIleIleLeuLeuLeuGlyLysAlaLeuSer                              195200205                                                                     ValValTrpAspArgHisLeuSerIleSerValValLeuLysGlnThr                              210215220                                                                     TyrGlnGluLysValCysGlyLeuCysGlyAsnPheAspGlyIleGln                              225230235240                                                                  AsnAsnAspLeuThrSerSerAsnLeuGlnValGluGluAspProVal                              245250255                                                                     AspPheGlyLysSerTrpGluValSerSerGlnCysAlaAspThrArg                              260265270                                                                     LysValProLeuAspSerSerProAlaThrCysHisAsnAsnIleMet                              275280285                                                                     LysGlnThrMetValAspSerSerCysArgIleLeuThrSerAspVal                              290295300                                                                     PheGlnAspCysAsnLysLeuValAspProGluProTyrLeuAspVal                              305310315320                                                                  CysIleTyrAspThrCysSerCysGluSerIleGlyAspCysAlaCys                              325330335                                                                     PheCysAspThrIleAlaAlaTyrAlaHisValCysAlaGlnHisGly                              340345350                                                                     LysValValThrTrpArgThrAlaThrLeuCysProGlnSerCysGlu                              355360365                                                                     GluArgAsnLeuArgGluAsnGlyTyrGluCysGluTrpArgTyrAsn                              370375380                                                                     SerCysAlaProAlaCysGlnValThrCysGlnHisProGluProLeu                              385390395400                                                                  AlaCysProValGlnCysValGluGlyCysHisAlaHisCysProPro                              405410415                                                                     GlyLysIleLeuAspGluLeuLeuGlnThrCysValAspProGluAsp                              420425430                                                                     CysProValCysGluValAlaGlyArgArgPheAlaSerGlyLysLys                              435440445                                                                     ValThrLeuAsnProSerAspProGluHisCysGlnIleCysHisCys                              450455460                                                                     AspValValAsnLeuThrCysGluAlaCysGlnGluProGlyGlyLeu                              465470475480                                                                  ValValProProThrAspAlaProValSerProThrThrLeuTyrVal                              485490495                                                                     GluAspIleSerGluProProLeuHisAspPheTyrCysSerArgLeu                              500505510                                                                     LeuAspLeuValPheLeuLeuAspGlySerSerArgLeuSerGluAla                              515520525                                                                     GluPheGluValLeuLysAlaPheValValAspMetMetGluArgLeu                              530535540                                                                     ArgIleSerGlnLysTrpValArgValAlaValValGluTyrHisAsp                              545550555560                                                                  GlySerHisAlaTyrIleGlyLeuLysAspArgLysArgProSerGlu                              565570575                                                                     LeuArgArgIleAlaSerGlnValLysTyrAlaGlySerGlnValAla                              580585590                                                                     SerThrSerGluValLeuLysTyrThrLeuPheGlnIlePheSerLys                              595600605                                                                     IleAspArgProGluAlaSerArgIleAlaLeuLeuLeuMetAlaSer                              610615620                                                                     GlnGluProGlnArgMetSerArgAsnPheValArgTyrValGlnGly                              625630635640                                                                  LeuLysLysLysLysValIleValIleProValGlyIleGlyProHis                              645650655                                                                     AlaAsnLeuLysGlnIleArgLeuIleGluLysGlnAlaProGluAsn                              660665670                                                                     LysAlaPheValLeuSerSerValAspGluLeuGluGlnGlnArgAsp                              675680685                                                                     GluIleValSerTyrLeuCysAspLeuAlaProGluAlaProProPro                              690695700                                                                     ThrLeuProProAspMetAlaGlnValThrValGlyProGlyLeuLeu                              705710715720                                                                  GlyValSerThrLeuGlyProLysArgAsnSerMetValLeuAspVal                              725730735                                                                     AlaPheValLeuGluGlySerAspLysIleGlyGluAlaAspPheAsn                              740745750                                                                     ArgSerLysGluPheMetGluGluValIleGlnArgMetAspValGly                              755760765                                                                     GlnAspSerIleHisValThrValLeuGlnTyrSerTyrMetValThr                              770775780                                                                     ValGluTyrProPheSerGluAlaGlnSerLysGlyAspIleLeuGln                              785790795800                                                                  ArgValArgGluIleArgTyrGlnGlyGlyAsnArgThrAsnThrGly                              805810815                                                                     LeuAlaLeuArgTyrLeuSerAspHisSerPheLeuValSerGlnGly                              820825830                                                                     AspArgGluGlnAlaProAsnLeuValTyrMetValThrGlyAsnPro                              835840845                                                                     AlaSerAspGluIleLysArgLeuProGlyAspIleGlnValValPro                              850855860                                                                     IleGlyValGlyProAsnAlaAsnValGlnGluLeuGluArgIleGly                              865870875880                                                                  TrpProAsnAlaProIleLeuIleGlnAspPheGluThrLeuProArg                              885890895                                                                     GluAlaProAspLeuValLeuGlnArgCysCysSerGlyGluGlyLeu                              900905910                                                                     GlnIleProThrLeuSerProAlaProAspCysSerGlnProLeuAsp                              915920925                                                                     ValIleLeuLeuLeuAspGlySerSerSerPheProAlaSerTyrPhe                              930935940                                                                     AspGluMetLysSerPheAlaLysAlaPheIleSerLysAlaAsnIle                              945950955960                                                                  GlyProArgLeuThrGlnValSerValLeuGlnTyrGlySerIleThr                              965970975                                                                     ThrIleAspValProTrpAsnValValProGluLysAlaHisLeuLeu                              980985990                                                                     SerLeuValAspValMetGlnArgGluGlyGlyProSerGlnIleGly                              99510001005                                                                   AspAlaLeuGlyPheAlaValArgTyrLeuThrSerGluMetHisGly                              101010151020                                                                  AlaArgProGlyAlaSerLysAlaValValIleLeuValThrAspVal                              1025103010351040                                                              SerValAspSerValAspAlaAlaAlaAspAlaAlaArgSerAsnArg                              104510501055                                                                  ValThrValPheProIleGlyIleGlyAspArgTyrAspAlaAlaGln                              106010651070                                                                  LeuArgIleLeuAlaGlyProAlaGlyAspSerAsnValValLysLeu                              107510801085                                                                  GlnArgIleGluAspLeuProThrMetValThrLeuGlyAsnSerPhe                              109010951100                                                                  LeuHisLysLeuCysSerGlyPheValArgIleCysMetAspGluAsp                              1105111011151120                                                              GlyAsnGluLysArgProGlyAspValTrpThrLeuProAspGlnCys                              112511301135                                                                  HisThrValThrCysGlnProAspGlyGlnThrLeuLeuLysSerHis                              114011451150                                                                  ArgValAsnCysAspArgGlyLeuArgProSerCysProAsnSerGln                              115511601165                                                                  SerProValLysValGluGluThrCysGlyCysArgTrpThrCysPro                              117011751180                                                                  CysValCysThrGlySerSerThrArgHisIleValThrPheAspGly                              1185119011951200                                                              GlnAsnPheLysLeuThrGlySerCysSerTyrValLeuPheGlnAsn                              120512101215                                                                  LysGluGlnAspLeuGluValIleLeuHisAsnGlyAlaCysSerPro                              122012251230                                                                  GlyAlaArgGlnGlyCysMetLysSerIleGluValLysHisSerAla                              123512401245                                                                  LeuSerValGluLeuHisSerAspMetGluValThrValAsnGlyArg                              125012551260                                                                  LeuValSerValProTyrValGlyGlyAsnMetGluValAsnValTyr                              1265127012751280                                                              GlyAlaIleMetHisGluValArgPheAsnHisLeuGlyHisIlePhe                              128512901295                                                                  ThrPheThrProGlnAsnAsnGluPheGlnLeuGlnLeuSerProLys                              130013051310                                                                  ThrPheAlaSerLysThrTyrGlyLeuCysGlyIleCysAspGluAsn                              131513201325                                                                  GlyAlaAsnAspPheMetLeuArgAspGlyThrValThrThrAspTrp                              133013351340                                                                  LysThrLeuValGlnGluTrpThrValGlnArgProGlyGlnThrCys                              1345135013551360                                                              GlnProIleLeuGluGluGlnCysLeuValProAspSerSerHisCys                              136513701375                                                                  GlnValLeuLeuLeuProLeuPheAlaGluCysHisLysValLeuAla                              138013851390                                                                  ProAlaThrPheTyrAlaIleCysGlnGlnAspSerSerHisGlnGlu                              139514001405                                                                  GlnValCysGluValIleAlaSerTyrAlaHisLeuCysArgThrAsn                              141014151420                                                                  GlyValCysValAspTrpArgThrProAspPheCysAlaMetSerCys                              1425143014351440                                                              ProProSerLeuValTyrAsnHisCysGluHisGlyCysProArgHis                              144514501455                                                                  CysAspGlyAsnValSerSerCysGlyAspHisProSerGluGlyCys                              146014651470                                                                  PheCysProProAspLysValMetLeuGluGlySerCysValProGlu                              147514801485                                                                  GluAlaCysThrGlnCysIleGlyGluAspGlyValGlnHisGlnPhe                              149014951500                                                                  LeuGluAlaTrpValProAspHisGlnProCysGlnIleCysThrCys                              1505151015151520                                                              LeuSerGlyArgLysValAsnCysThrThrGlnProCysProThrAla                              152515301535                                                                  LysAlaProThrCysGlyLeuCysGluValAlaArgLeuArgGlnAsn                              154015451550                                                                  AlaAspGlnCysCysProGluTyrGluCysValCysAspProValSer                              155515601565                                                                  CysAspLeuProProValProHisCysGluArgGlyLeuGlnProThr                              157015751580                                                                  LeuThrAsnProGlyGluCysArgProAsnPheThrCysAlaCysArg                              1585159015951600                                                              LysGluGluCysLysArgValSerProProSerCysProProHisArg                              160516101615                                                                  LeuProThrLeuArgLysThrGlnCysCysAspGluTyrGluCysAla                              162016251630                                                                  CysAsnCysValAsnSerThrValSerCysProLeuGlyTyrLeuAla                              163516401645                                                                  SerThrAlaThrAsnAspCysGlyCysThrThrThrThrCysLeuPro                              165016551660                                                                  AspLysValCysValHisArgSerThrIleTyrProValGlyGlnPhe                              1665167016751680                                                              TrpGluGluGlyCysAspValCysThrCysThrAspMetGluAspAla                              168516901695                                                                  ValMetGlyLeuArgValAlaGlnCysSerGlnLysProCysGluAsp                              170017051710                                                                  SerCysArgSerGlyPheThrTyrValLeuHisGluGlyGluCysCys                              171517201725                                                                  GlyArgCysLeuProSerAlaCysGluValValThrGlySerProArg                              173017351740                                                                  GlyAspSerGlnSerSerTrpLysSerValGlySerGlnTrpAlaSer                              1745175017551760                                                              ProGluAsnProCysLeuIleAsnGluCysValArgValLysGluGlu                              176517701775                                                                  ValPheIleGlnGlnArgAsnValSerCysProGlnLeuGluValPro                              178017851790                                                                  ValCysProSerGlyPheGlnLeuSerCysLysThrSerAlaCysCys                              179518001805                                                                  ProSerCysArgCysGluArgMetGluAlaCysMetLeuAsnGlyThr                              181018151820                                                                  ValIleGlyProGlyLysThrValMetIleAspValCysThrThrCys                              1825183018351840                                                              ArgCysMetValGlnValGlyValIleSerGlyPheLysLeuGluCys                              184518501855                                                                  ArgLysThrThrCysAsnProCysProLeuGlyTyrLysGluGluAsn                              186018651870                                                                  AsnThrGlyGluCysCysGlyArgCysLeuProThrAlaCysThrIle                              187518801885                                                                  GlnLeuArgGlyGlyGlnIleMetThrLeuLysArgAspGluThrLeu                              189018951900                                                                  GlnAspGlyCysAspThrHisPheCysLysValAsnGluArgGlyGlu                              1905191019151920                                                              TyrPheTrpGluLysArgValThrGlyCysProProPheAspGluHis                              192519301935                                                                  LysCysLeuAlaGluGlyGlyLysIleMetLysIleProGlyThrCys                              194019451950                                                                  CysAspThrCysGluGluProGluCysAsnAspIleThrAlaArgLeu                              195519601965                                                                  GlnTyrValLysValGlySerCysLysSerGluValGluValAspIle                              197019751980                                                                  HisTyrCysGlnGlyLysCysAlaSerLysAlaMetTyrSerIleAsp                              1985199019952000                                                              IleAsnAspValGlnAspGlnCysSerCysCysSerProThrArgThr                              200520102015                                                                  GluProMetGlnValAlaLeuHisCysThrAsnGlySerValValTyr                              202020252030                                                                  HisGluValLeuAsnAlaMetGluCysLysCysSerProArgLysCys                              203520402045                                                                  SerLys                                                                        2050                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 681 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..681                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGTTGCACGATTTCTACTGCAGCAGGCTACTGGACCTGGTCTTCCTG48                            MetLeuHisAspPheTyrCysSerArgLeuLeuAspLeuValPheLeu                              151015                                                                        CTGGATGGCTCCTCCAGGCTGTCCGAGGCTGAGTTTGAAGTGCTGAAG96                            LeuAspGlySerSerArgLeuSerGluAlaGluPheGluValLeuLys                              202530                                                                        GCCTTTGTGGTGGACATGATGGAGCGGCTGCGCATCTCCCAGAAGTGG144                           AlaPheValValAspMetMetGluArgLeuArgIleSerGlnLysTrp                              354045                                                                        GTCCGCGTGGCCGTGGTGGAGTACCACGACGGCTCCCACGCCTACATC192                           ValArgValAlaValValGluTyrHisAspGlySerHisAlaTyrIle                              505560                                                                        GGGCTCAAGGACCGGAAGCGACCATCAGAGCTGCGGCGCATTGCCAGC240                           GlyLeuLysAspArgLysArgProSerGluLeuArgArgIleAlaSer                              65707580                                                                      CAGGTGAAGTATGCGGGCAGCCAGGTGGCCTCCACCAGCGAGGTCTTG288                           GlnValLysTyrAlaGlySerGlnValAlaSerThrSerGluValLeu                              859095                                                                        AAATACACACTGTTCCAAATCTTCAGCAAGATCGACCGCCCTGAAGCC336                           LysTyrThrLeuPheGlnIlePheSerLysIleAspArgProGluAla                              100105110                                                                     TCCCGCATCGCCCTGCTCCTGATGGCCAGCCAGGAGCCCCAACGGATG384                           SerArgIleAlaLeuLeuLeuMetAlaSerGlnGluProGlnArgMet                              115120125                                                                     TCCCGGAACTTTGTCCGCTACGTCCAGGGCCTGAAGAAGAAGAAGGTC432                           SerArgAsnPheValArgTyrValGlnGlyLeuLysLysLysLysVal                              130135140                                                                     ATTGTGATCCCGGTGGGCATTGGGCCCCATGCCAACCTCAAGCAGATC480                           IleValIleProValGlyIleGlyProHisAlaAsnLeuLysGlnIle                              145150155160                                                                  CGCCTCATCGAGAAGCAGGCCCCTGAGAACAAGGCCTTCGTGCTGAGC528                           ArgLeuIleGluLysGlnAlaProGluAsnLysAlaPheValLeuSer                              165170175                                                                     AGTGTGGATGAGCTGGAGCAGCAAAGGGACGAGATCGTTAGCTACCTC576                           SerValAspGluLeuGluGlnGlnArgAspGluIleValSerTyrLeu                              180185190                                                                     TGTGACCTTGCCCCTGAAGCCCCTCCTCCTACTCTGCCCCCCGACATG624                           CysAspLeuAlaProGluAlaProProProThrLeuProProAspMet                              195200205                                                                     GCACAAGTCACTGTGGGCCCGGGGCTCTTGGGGGTTTCGACCCTGGGG672                           AlaGlnValThrValGlyProGlyLeuLeuGlyValSerThrLeuGly                              210215220                                                                     CCCAAGTAA681                                                                  ProLys                                                                        225                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 226 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLeuHisAspPheTyrCysSerArgLeuLeuAspLeuValPheLeu                              151015                                                                        LeuAspGlySerSerArgLeuSerGluAlaGluPheGluValLeuLys                              202530                                                                        AlaPheValValAspMetMetGluArgLeuArgIleSerGlnLysTrp                              354045                                                                        ValArgValAlaValValGluTyrHisAspGlySerHisAlaTyrIle                              505560                                                                        GlyLeuLysAspArgLysArgProSerGluLeuArgArgIleAlaSer                              65707580                                                                      GlnValLysTyrAlaGlySerGlnValAlaSerThrSerGluValLeu                              859095                                                                        LysTyrThrLeuPheGlnIlePheSerLysIleAspArgProGluAla                              100105110                                                                     SerArgIleAlaLeuLeuLeuMetAlaSerGlnGluProGlnArgMet                              115120125                                                                     SerArgAsnPheValArgTyrValGlnGlyLeuLysLysLysLysVal                              130135140                                                                     IleValIleProValGlyIleGlyProHisAlaAsnLeuLysGlnIle                              145150155160                                                                  ArgLeuIleGluLysGlnAlaProGluAsnLysAlaPheValLeuSer                              165170175                                                                     SerValAspGluLeuGluGlnGlnArgAspGluIleValSerTyrLeu                              180185190                                                                     CysAspLeuAlaProGluAlaProProProThrLeuProProAspMet                              195200205                                                                     AlaGlnValThrValGlyProGlyLeuLeuGlyValSerThrLeuGly                              210215220                                                                     ProLys                                                                        225                                                                           __________________________________________________________________________

What is claimed is:
 1. A non-glycosylated, biologically activepolypeptide consisting of the amino acid sequence: ##STR11## wherein Xis NH₂ -methionine- or NH₂ -- and the two cysteines included in thesequence are joined by a disulfide bond.
 2. A pharmaceutical compositioncomprising an amount of a polypeptide of claim 1 effective to inhibitplatelet aggregation and a pharmaceutically acceptable carrier.
 3. Amethod of inhibiting platelet aggregation which comprises contactingplatelets with an amount of a polypeptide of claim 1 effective toinhibit platelet aggregation so as to inhibit platelet aggregation.
 4. Amethod of claim 3 wherein the amount effective to inhibit plateletaggregation is 0.1-200 mg/kg body weight.
 5. A method of claim 4 whereinthe amount effective to inhibit platelet aggregation is 1-20 mg/kg bodyweight.
 6. A method of claim 3 wherein the amount effective to inhibitplatelet aggregation is an amount sufficient to maintain a bloodconcentration of 0.1-10 μM polypeptide.
 7. A method of claim 6 whereinthe blood concentration is maintained at about 1 μM polypeptide.
 8. Amethod of treating a subject with a cerebrovascular disorder whichcomprises administering to the subject an amount of a polypeptide ofclaim 1 effective to inhibit platelet aggregation.
 9. A method oftreating a subject with a cardiovascular disorder which comprisesadministering to the subject an amount of a polypeptide of claim 1effective to inhibit platelet aggregation.
 10. A method of treating asubject in accordance with claim 9, wherein the cardiovascular disordercomprises acute myocardial infarction.
 11. A method of treating asubject in accordance with claim 9, wherein the cardiovascular disordercomprises angina.
 12. A method of inhibiting platelet aggregation in asubject prior to, during, or after the subject has undergoneangioplasty, thrombolytic treatment, or coronary bypass surgery whichcomprises administering to the subject an amount of a polypeptide ofclaim 1 effective to inhibit platelet aggregation.
 13. A method ofmaintaining blood vessel patency in a subject prior to, during, or afterthe subject has undergone coronary bypass surgery, which comprisesadministering to the subject an amount of a polypeptide of claim 1effective to inhibit platelet aggregation.
 14. A method of inhibitingthrombosis in a subject which comprises administering to the subject anamount of a polypeptide of claim 1 effective to inhibit the thrombosis.15. A method of claim 14, wherein the thrombosis is associated with aninflammatory response.
 16. A polypeptide in accordance with claim 1bound to a solid matrix.
 17. A method of treating a subject sufferingfrom platelet adhesion to damaged vascular surfaces which comprisesadministering to the subject an amount of the polypeptide of claim 1effective to inhibit platelet adhesion to damaged vascular surfaces. 18.A method of claim 17 wherein the amount effective to inhibit plateletadhesion is 0.1-200 mg/kg body weight.
 19. A method of claim 18 whereinthe amount effective to inhibit platelet aggregation is 1-20 mg/kg bodyweight.
 20. A method of claim 17 wherein the amount effective to inhibitplatelet adhesion is the amount sufficient to maintain a bloodconcentration of 0.1-10 μM polypeptide.
 21. A method of claim 20 whereinthe blood concentration is maintained at about 1 μM polypeptide.
 22. Amethod of thrombolytic treatment of thrombi-containing, platelet-richaggregates in a subject which comprises administering to the subject anamount of the polypeptide of claim 1 effective to treatthrombi-containing, platelet-rich aggregates.
 23. A method of preventingplatelet adhesion to a prosthetic material or device in a subject whichcomprises administering to the subject an amount of the polypeptide ofclaim 1 effective to prevent platelet adhesion to the material ordevice.
 24. A method of inhibiting re-occlusion in a subject followingangioplasty or thrombolysis which comprises administering to the subjectan amount of the polypeptide of claim 1 effective to inhibitre-occlusion.